Lentiviral vectors for delivery of pklr to treat pyruvate kinase deficiency

ABSTRACT

The present disclosure provides polynucleotide cassettes, expression vectors and methods for the expression of a gene in mammalian cells to provide gene therapy for pyruvate kinase deficiency.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is continuation of U.S. patent application Ser. No.16/755,033, which is a National Stage application under 35 U.S.C. § 371of International Application No. PCT/US2018/056136, filed Oct. 16, 2018,which claims the benefit of U.S. Provisional Application No. 62/573,037,filed on Oct. 16, 2017, each of which is incorporated by referenceherein in their entirety.

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FIELD OF THE INVENTION

This invention pertains to gene therapy of Pyruvate Kinase Deficiency.

BACKGROUND OF THE INVENTION

Pyruvate Kinase Deficiency (PKD) is a monogenic metabolic disease causedby mutations in the PKLR gene that leads to hemolytic anemia of variablesymptomatology and that can be fatal during the neonatal period. PKDrecessive inheritance trait and its curative treatment by allogeneicbone marrow transplantation provide an ideal scenario for developinggene therapy approaches.

Among many other hereditary enzymatic defects affecting theerythrocytes, Pyruvate Kinase deficiency (PKD) is the most frequent onecausing chronic nonspherocytic hemolytic anemia (CNSHA) (Zanella et al.2007). Onset and severity of PKD are very variable and range from mildto severe neonatal anemia, becoming fatal during the childhood in themost severe cases (Pissard et al. 2006). Growth retardation, hydropsfetalis and death during the neonatal period have also been reportedwith low frequency (Gilsanz et al. 1993). PKD prevalence has beenestimated at 1:20,000 in the general Caucasian population (Beutler etal. 2000) and, so far, more than 195 different mutations in the PKLRgene have been identified (http://www.lovd.nl/pklr).

Allogeneic bone marrow transplantation (BMT) has been successfully usedto cure severe PKD patients (Tanphaichitr et al. 2000), but the lowavailability of histocompatible donors and the serious complicationsassociated with the BMT of these patients (i.e. graft versus hostdisease, opportunistic infections, etc.) make periodic bloodtransfusions and splenectomy the main therapeutic options for most ofthe severe forms of PKD (Zanella et al. 2005), dramatically increasingpatient morbidity and mortality (Hilgard et al. 2005). The limitedefficacy and side effects of the therapeutic options for severe PKDpatients and its recessive inheritance trait make PKD a suitable diseaseto be treated by gene therapy.

PKD is caused by defects in the Pyruvate Kinase (PK) enzyme (Zanella2005) that catalyses the last ATP-generating reaction of the glycolysispathway in all cells. In mature erythrocytes PK becomes essential asRBCs only express the R-type specific isoform (RPK) (Kanno et al. 1992)due to the regulation of the erythroid specific alternative promoter ofthe PKLR locus (Noguchi et al. 1987). Thus, any loss of RPK activityimpairs RBC metabolism and lifespan (Zanella 2005), leading to CNSHA.

A promising approach to treating and preventing genetic and otherdiseases and disorders is delivery of therapeutic agents with a genetherapy vector. Currently, viral vectors show the greatest efficiency ingene transfer, and for correction of genetic diseases such thatpersistent gene expression is required, herpesvirus, retrovirus,lentivirus, adenovirus, or AAV based vectors are desirable due to theintegrating nature of the viral life cycle.

Gene therapy for monogenic diseases, particularly those affecting thehematopoietic system, has provided convincing evidence that geneticcorrection of autologous hematopoietic stem cells (HSCs) is analternative therapeutic option to allogeneic HSCT, avoiding its majorcomplications (Cartier et al 2009; Cavazzana-Calvo et al 2010; Cartieret al 2012; Aiuti et al 2013; Biffi et al 2013). Genetic correction fordiseases affecting the erythrocyte such as β-thalassemia and sickle celldisease, have been addressed in animal models (Pestina et al 20091 Bredaet al 2012) and also in humans (Cavazzana-Calvo et al 2010). However,gene therapy approaches for inherited erythroid metabolic deficienciessuch as PKD are still limited.

The feasibility of HSC gene therapy for PKD has been demonstrated bothin mouse (Tani et al 1994; Meza et al 2009) and in dog RPK deficientexperimental models (Trobridge et al 2012) showing that donor chimerismor level of gene-modified cells are key points to reach an efficientcorrection of the hemolytic phenotype (Richard et al 2004) given thelack of selective advantage of gene-corrected HSCs. Previous work with aPKD mouse model demonstrated that retrovirally-derived human RPKexpression was capable of fully correcting PKD phenotype when over 25%genetically corrected cells were transplanted (Meza et al 2009). Asimilar therapeutic threshold of corrected cells was recently reportedin one PKD Basenji dog infused with in vivo expanded and foamyvector-corrected HSCs (Trobridge et al 2012).

A number of challenges remain with regard to designing polynucleotidecassettes and expression vectors for use in gene therapy. Onesignificant challenge is obtaining sufficient expression of thetransgene in target cells. A longstanding unmet need in the art has beensufficiently robust expression of transgenes following gene transfer. Insome cases, more efficient expression is required for the efficacy ofcertain vectors, for example plasmid DNA vectors. In other cases, moreefficient gene expression cassettes are desirable to allow for a lowertherapeutic dose that has a more favorable safety profile or a lessinvasive route of administration.

High levels of transgene expression can be achieved when gammaretroviral (gamma-RV) vectors are used due to the fact that thetherapeutic transgene expression is regulated by their long terminalrepeats (LTR). However, the first clinical trials based on this type ofvector raised safety concerns, as several patients developed unexpectedleukemias (Hacein-Bey-Abina et al 2008). The strong promoter activity ofLTRs could affect the regulation of surrounding genes, either byactivation of proto-oncogene promoters or by inhibition of tumoursuppressor genes, leading to insertional mutagenesis (Ott et al 2006;Howe et al 2008; Stein et al 2010; Braun et al 2014). These findingshighlighted the need to use safer and more efficient vectors thangamma-RV vectors for the PKD gene therapy.

SUMMARY OF THE INVENTION

In one embodiment, the present invention provides an expression cassettecomprising a polynucleotide sequence comprising: a) a promoter sequence;b) a sequence encoding a gene product; and c) a ribonucleic acid (RNA)export signal, wherein the promoter sequence is operably linked to thesequence encoding the pyruvate kinase polypeptide, and optionally wherea)-c) are present in the expression cassette in 5′ to 3′ order. Incertain embodiments, the promoter is a phosphoglycerate kinase (PGK)promoter. In some embodiments, the gene product is a therapeutic geneproduct. In some embodiments, the therapeutic gene product is a pyruvatekinase (PK) polypeptide, optionally a pyruvate kinase, liver and redblood cell (PKLR) polypeptide. In certain embodiments, the sequenceencoding the gene product is codon-optimized. In some embodiments, thesequence encoding the gene product is a codon-optimized version of thehuman pyruvate kinase cDNA having at least 85% identity to SEQ ID NO:8.In particular embodiments, the RNA export signal is a mutatedpost-transcriptional regulatory element of the woodchuck hepatitis virus(wPRE).

In certain embodiments, the mutated wPRE is a chimeric wPRE comprising asequence having at least 80% identity to SEQ ID NO:24. In someembodiments, the expression cassette further comprising one or moreenhancer sequences. In some embodiments, the expression cassette furthercomprises a polypurine tract (PPT) or polyadenylation (polyA) signalsequence. In some embodiments, the expression cassette further comprisesone or more of the following sequences: i) a packing signal sequence;ii) a truncated Gag sequence; iii) a Rev responsive element (RRE); iv) acentral polypurine tract (cPPT); v) a central terminal sequence (CTS);and vi) an upstream sequence element (USE), optionally from simian virus40 (SV40-USE). In some embodiments, the expression cassette furthercomprises 5′ and 3′ long terminal repeat (LTR) sequences.

In a related embodiment, the present invention provides a recombinantgene delivery vector comprising an expression cassette disclosed herein.In certain embodiments, the recombinant gene delivery vector is a virusor viral vector. In certain embodiments, the virus or viral vector is alentivirus (LV).

In another related embodiment, the present invention provides a cellcomprising an expression cassette or gene delivery vector disclosedherein. In some embodiments, the cell is a blood cell. In someembodiments, the cell is an erythroid cell. In some embodiments, thecell is a bone marrow cell, e.g., a lineage depleted bone marrow cell.In some embodiments, the cell is a hematopoietic stem cell. In someembodiments, the cell is a CD34+ hematopoietic stem cell. In someembodiments, the cell is a committed hematopoietic erythroid progenitorcell.

In yet another related embodiment, the present invention provides apharmaceutical composition comprising a pharmaceutically acceptableexcipient and recombinant gene delivery vector or cell disclosed herein.

In another embodiment, the present invention provides a method oftreating or preventing a disease or disorder in a subject in needthereof, comprising providing to the subject an expression cassette,gene delivery vector, or pharmaceutical composition disclosed herein. Inone embodiment, the disease or disorder is a PKD and the gene product isa PK polypeptide, optionally a PKLR polypeptide. In certain embodiments,the pharmaceutical composition comprises the recombinant gene deliveryvector. In other embodiments, the pharmaceutical composition comprisesthe cell. In one embodiment, the cell is autologous to the subject.

In a related embodiment, the present invention provides a method forexpressing a transgene in erythroid cells, comprising contacting one ormore erythroid cells with an effective amount of a recombinant viralvector, wherein the vector comprises a human PGK promoter, a codonoptimized version of a human PKLR cDNA transgene, and a mutated wPRE,wherein following said contacting, PKLR is expressed at detectablelevels in the one or more erythroid cells.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the disclosure are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 depicts a scheme reflecting the position of different elementspresent in the backbone of an illustrative LV vector.

FIG. 2A is a schematic representation of illustrative self-inactivating(SIN) LV vectors used throughout gene therapy experiments harboring thehuman PGK promoter regulating the expression of the EGFP transgene inthe control vector (upper diagram) or the expression of acodon-optimized sequence of the PKLR gene cDNA (coRPK) in thetherapeutic vector (lower diagram).

FIG. 2B is a schematic of a gene therapy protocol performed to addressthe functionality of the developed PGK-coRPK LV vector.

FIGS. 3A-3D depict data showing correction of PKD phenotype inperipheral blood of primary recipients after genetic correction. FIGS.3A and 3B show RBC and reticulocyte levels, respectively, in healthy(black bar, n=5) and PKD anemic mice (gray bar, n=6), and PKD anemicmice that were transplanted with the EGFP (white bar, n=9) or coRPKtransduced cells (scratched bar, n=17). Data are represented as theaverage±SEM and were analyzed by non-parametric Kruskal-Wallis test.FIG. 3C shows the flow cytometry strategy used to detect the biotinlabelled RBCs throughout the time and FIG. 3D shows RBC survivalkinetics in healthy (black line, n=2), anemic (gray line, n=2) andgenetically corrected mice (discontinuous line, n=4). Data arerepresented as the average±SEM and were analyzed by two-way ANOVA test.Healthy, non-transplanted control mice; PKD, non-transplanted PKD mice;coRPK, PKD mice expressing the therapeutic transgene.

FIGS. 4A-4C show multi-lineage hematopoietic reconstitution in secondarytransplanted mice. FIG. 4A is a diagram of the flow cytometry strategyused to identify the different hematopoietic lineages by labeling withCD3-PE, B220-PE, B220-PECy5, Grl-Biotin and Macl-Biotin antibodies plusSAV-PE-Cy5. FIG. 4B depicts representative dot-plots and FIG. 4C depictspercentages of each lineage in PB at 140 days after transplant. Barsrepresent the average percentage±SEM of healthy (n=2, black bar) and PKDmouse (n=2, grey bar) controls and secondary transplanted miceexpressing the coRPK therapeutic transgene (n=4, scratched bar).

FIGS. 5A-5D depict PKD phenotype correction in secondary transplantedmice. FIG. 5A shows Brilliant Cresyl blue staining of blood smears fromnon-transplanted mice and secondary recipients to identify reticulocytepopulation (in blue). FIG. 5B is flow cytometry analysis of reticulocytelevels in peripheral blood. FIG. 5C represents RBC percentage and FIG.5D represents reticulocyte percentage in secondary transplanted miceexpressing the coRPK transgene (scratched bar, n=4), in healthy mice(black bar, n=3) and in anemic control mice (grey bar, n=3). Data arerepresented the average±SEM and were analyzed by non-parametrictwo-tailed Mann-Whitney test.

FIGS. 6A-6C show quantification of proviral integrations. FIG. 6A showsvector copy number per cell in BM CFUs from individual transplanted miceat 120 to 170 days after transplant. Transduction and chimerismpercentages are also shown. FIG. 6B shows provirus copy number in cellsfrom different hematopoietic compartments. Columns represent theaverage±SEM of the different groups of transplanted mice. FIG. 6C showsthe kinetics of proviral integrations in BM cells from individualtransplanted EGFP-expressing mice (grey lines) and mice carrying thecoRPK transgene (black lines).

FIGS. 7A-7C depict the normalization of the erythroid differentiationpattern in genetically corrected mice. FIG. 7A shows percentages of thedifferent erythroid subpopulation in bone marrow and spleen at 140 daysafter transplant. FIG. 7B depicts representative dot plots of the flowcytometry strategy used. The expression intensity of the CD71 and Ter119markers allows for identifying four erythroid subpopulations. populationI: early proerythroblasts (Ter119^(med) CD71^(high)), population II:basophilic erythroblasts (Ter119^(high) CD71^(high)), population III:late basophilic and polychromatophilic erythroblasts (Ter119^(high)CD71^(med)) and population IV: orthochromatophilic erythroblasts,reticulocytes and mature erythroid cells (Ter119^(high) CD71^(low)).FIG. 7C shows plasma Epo levels measured by ELISA in non-transplantedand transplanted mice. Dots represent values of individual mice. Linesrepresent average±SEM and were analyzed by non-parametric Kruskal-Wallistest. Healthy, non-transplanted control mice; PKD, non-transplanted PKDmice; EGFP, PKD mice expressing the EGFP transgene; coRPK, PKD miceexpressing the therapeutic transgene.

FIGS. 8A-8B show hematopoietic progenitor assays in control mice andtransplanted mice with transduced cells. The data demonstrate total CFUsfrom spleen (FIG. 8A) and bone marrow (FIG. 8B) at 140 days aftertransplant. Dots represent number of colonies per mouse analyzed andlines represent average±SEM in each group. Data were statisticallyanalyzed by non-parametric Kruskal-Wallis test.

FIGS. 9A-9C demonstrate reversion of splenomegaly and organ pathology ingenetically corrected mice at 140 days post-transplantation. FIG. 9Ashows pictures of representative spleens, and FIG. 9B shows ratio ofspleen weight to total body weight from primary and secondarytransplanted PKD mice. Dots represent values of individual mice. Linesrepresent average±SEM per group. Data were analyzed by non-parametricKruskal-Wallis test. FIG. 9C shows the histological study of spleen andliver from primary transplanted PKD mice. First and second column showthe representative histology sections of spleen and liver stained withhematoxylin-eosin and photographed using a 4× and 10× objective,respectively, in a light microscope. Arrows point to erythroid cellclusters indicative of extramedullary erythropoiesis. Third column showsPrussian blue staining (Fe) of liver sections to detect iron depositsindicated by arrowheads. Photographs were taken using a 20× objective.Group legends as in FIG. 7 . 2^(nd) coRPK, secondary recipients.

FIGS. 10A-10G depict metabolic profiling in RBC samples from micetransplanted with genetically modified cells. Analysis of significantmetabolic profile changes in healthy and transplanted mice by comparisonto PKD animals in two independent experiments. FIG. 10A shows thecomplete RBC heat map obtained by untargeted profiling, where higher andlower metabolite levels are represented in red and blue respectively.Metabolites listed have at least one comparison that is significantusing the following criteria: absolute fold change>1.5; minimalsignal>2000; Adjusted p-value<0.01. Black boxes highlight cluster ofmetabolite changes with distinct profile among the groups. FIGS. 10B,10C, and 10D depict ATP, ADP and pyruvate levels in RBCs, respectively,measured by untargeted profiling by comparison to PKD mice at 140 daysafter transplant. Assay 1: Healthy mice (black bars) n=1, PKD (greybars) n=1, hPGK-EGFP (white bars) n=2, hPGK-coRPK (scratched bars) n=3.Assay 2: Healthy mice n=2, PKD n=2, hPGK-EGFP n=6, hPGK-coRPK n=10.FIGS. 10Ee, 10F, and 10G depict RBC targeted metabolic profiling of aselected number of metabolites involved in the glycolytic pathway (PEP,3-phosphoglyceric acid and D-lactic acid, respectively) at 280 dayspost-transplantation. Dots represent values of individual mice. Linesrepresent average±SEM and were analyzed by non-parametric Kruskal-Wallistest. Assay 2: Healthy mice n=7, PKD n=5, hPGK-EGFP n=3, hPGK-coRPK n=5.

FIGS. 11A-11C depict pyruvate kinase activity, hexokinase activity andratio of pyruvate kinase and hexokinase enzymatic activities,respectively, in RBCs from control mice and mice transplanted withtransduced cells. RBCs were purified from blood samples through acellulose column to avoid leukocyte PK activity contamination andsubjected to enzyme activity evaluation. Black bars, healthy mice (n=2);white bars, mice transplanted with cells transduced with the EGFPexpressing vector (n=3); scratched bars, mice transplanted with cellstransduced with the coRPK expressing vector (n=3). Checkered barsrepresent values from a healthy volunteer (n=1). Data represent theaverage±SEM of each group.

FIGS. 12A-12D show untargeted metabolic profiling in WBC samples frommice transplanted with genetically modified cells. FIG. 12A representsthe principal component analysis of untargeted metabolite profile inRBCs (red dots; left and center) and WBCs (blue dots; cluster on right)in control and transplanted mice. FIGS. 12B, 12C, and 12D depict ATP,ADP and pyruvate levels, respectively, in WBCs by comparison to PKDmice. Assay 1: healthy mice (black bars) n=1, PKD (grey bars) n=1,hPGK-EGFP (white bars) n=2, hPGK-coRPK (scratched bars) n=3. Assay 2:healthy mice n=2, PKD n=2, hPGK-EGFP n=6, hPGK-coRPK n=10. Datarepresent the average±SEM per group and were analyzed by non-parametricKruskal-Wallis test.

FIG. 13 shows a gel image of LAM-PCR products generated with Tsp509Ienzyme for samples harvested from all mice at different time points andtissues. Vector integration sites were identified by LAM-PCRamplification of 3′vector LTR-genome junctions. A MultiNA automatedsystem was used, generating a pattern characterized by several bands.Vector backbone derived Tsp509I internal control band (IC) is indicatedby an arrow.

FIG. 14 shows a gel image of LAM-PCR products generated with HpyCH4IV5enzyme for samples harvested from all mice at different time points andtissues. Vector integration sites were identified by LAM-PCRamplification of 3′ vector LTR-genome junctions. A MultiNA automatedsystem was used, generating a pattern characterized by several bands.Vector backbone derived HpyCH4IV5 IC is indicated by an arrow.

FIG. 15 depicts a general scheme of the analysis of integration sitemapping performed in mice transplanted with genetically modifiedhematopoietic progenitors. Bone marrow and white blood cell samples fromtransplanted mice belonging to two independent experiments (Table 3) andharvested at different time-points after transplant were analyzed asdescribed in supplementary methods following the showed pipeline.

FIGS. 16A-16B show the distribution of LV integrations along the genomeof transplanted mice. FIG. 16A depicts integration site (IS) frequencydistribution around Transcription Start Site (TSS) of the nearest RefSeqgene, spanning 500 Kb upstream and downstream the TSS. Numbers on thetop are the number of IS detected for all samples and time-points. FIG.16B depicts chromosomal distribution of LV integration sites intransplanted mice expressing the EGFP transgene (black bars) or thecoRPK therapeutic transgene (grey bars), showing no skewing towards anyparticular chromosome.

FIGS. 17A-17C demonstrates clonal abundance analysis of coRPK-LVtransduced cells. Dots plot representation of clonal abundance of pooledintegrations in each mouse from assays 1 (FIG. 17A) and 2 (FIGS. 17B and17C). The relative percentage (y-axis) for each IS is relative to thetotal number of sequences reads obtained in each dataset. BM, bonemarrow; PB, peripheral blood; coRPK1-14, mice transplanted withhematopoietic cells transduced with the therapeutic vector.

FIG. 18 presents the tracked shared integrations between primary andsecondary recipient mice carrying the therapeutic PGK-coRPK LV vector.Integrations detected in either mouse in any organ and at any time arepooled. Secondary recipients received the pooled BM from transplantedmice coRPK11 to 14. The rest of the IS detected were detected or in theprimary or in the secondary recipients. Numbers in the boxes show therepresentativeness in percentage of the corresponding integration in thereferred mouse. In addition to ≥5% filter applied on integrationanalysis, all integration with a sequence count <3 were eliminated.

FIG. 19 demonstrates clonal abundance analysis of EGFP-LV transducedcells. Dots plot representation of clonal abundance of pooledintegrations in each mouse in bone marrow. The relative percentage(y-axis) for each IS is relative to the total number of sequences readsobtained in each dataset. Similarly to co-RPK transduced cells (FIG. 17), the graph indicates that the vast majority of transplanted mice showa polyclonal pattern of hematopoietic repopulation.

FIG. 20 depicts the LV genomic integration profile. Gene Ontology (GO)analysis was performed using the GREAT software on samples fromtransplanted mouse. All integrations retrieved from this study (N=2220)showed overrepresentations of the gene functions indicated on the leftpart of the figure. To address if the most abundant integrations wereenriched on specific gene classes, all ISs with a relative sequencecount >5% of the entire dataset (shown in FIG. 17 ) were selected,showing no GO gene classes overrepresented.

FIG. 21 depicts a schematic diagram of the PGK-coRPK LV lentiviralvector.

FIG. 22 depicts a diagram of a plasmid (CPcoRPKW-17) comprising thePGK-coRPK LV lentiviral vector.

FIGS. 23A-23F represent the nucleotide sequence of the PGK-coRPK LVlentiviral vector, annotated to show the location of the variousfunctional elements.

FIGS. 24A-24B depicts the mechanism of action of the PGK-coRPK LVlentiviral vector. FIG. 24A depicts the ectopic expression of thePGK-coRPK LV vector will rescue the wild-type phenotype of PKDerythrocytes, otherwise unable to generate a functional RPK protein toproduce sufficient energy to carry out their functions. FIG. 24B showsthe gene therapy strategy for PKD patients based on the ex vivotransduction of RPK deficient CD34⁺ hematopoietic progenitors with thePGK-coRPK LV lentiviral vector and subsequent transplantation into thepatient. The developed PGK-coRPK LV lentiviral vector carrying thetherapeutic human PKLR gene cDNA will be integrated in the patient CD34⁺cell genome upon ex vivo transduction. These genetically corrected cellswill be then infused back into the patient, where they will produce RBCsexpressing the therapeutic transgene, and therefore, producingfunctional RPK proteins that will correct the PKD pathologicalphenotype. Figure modified from the Boston Children's Hospital blog.

DETAILED DESCRIPTION OF THE INVENTION Definitions

A “vector” as used herein refers to a macromolecule or association ofmacromolecules that comprises or associates with a polynucleotide andwhich can mediate delivery of the polynucleotide to a cell. Illustrativevectors include, for example, plasmids, viral vectors, liposomes, andother gene delivery vehicles.

The term “LV” is an abbreviation for lentivirus, and may refer to thevirus itself or derivatives thereof. The term covers all subtypes andboth naturally occurring and recombinant forms, except where requiredotherwise.

As used herein, the term “gene” or “coding sequence” refers to anucleotide sequence in vitro or in vivo that encodes a gene product. Insome instances, the gene consists or consists essentially of codingsequence, that is, sequence that encodes the gene product. In otherinstances, the gene comprises additional, non-coding, sequence. Forexample, the gene may or may not include regions preceding and followingthe coding region, e.g. 5′ untranslated (5′ UTR) or “leader” sequencesand 3′ UTR or “trailer” sequences, as well as intervening sequences(introns) between individual coding segments (exons).

As used herein, a “therapeutic gene” refers to a gene that, whenexpressed, confers a beneficial effect on the cell or tissue in which itis present, or on a mammal in which the gene is expressed. Examples ofbeneficial effects include amelioration of a sign or symptom of acondition or disease, prevention or inhibition of a condition ordisease, or conferral of a desired characteristic. Therapeutic genesinclude genes that correct a genetic deficiency in a cell or mammal.

As used herein, a transgene is a gene delivered to a cell by a vector.

As used herein, the term “gene product” refers to the desired expressionproduct of a polynucleotide sequence such as a polypeptide, peptide,protein or interfering RNA including short interfering RNA (siRNA),miRNA or small hairpin RNA (shRNA).

As used herein, the terms “polypeptide,” “peptide,” and “protein” referto polymers of amino acids of any length. The terms also encompass anamino acid polymer that has been modified; for example, disulfide bondformation, glycosylation, lipidation, phosphorylation, or conjugationwith a labeling component.

By “comprising” it is meant that the recited elements are required in,for example, the composition, method, kit, etc., but other elements maybe included to form the, for example, composition, method, kit etc.within the scope of the claim. For example, an expression cassette“comprising” a gene encoding a therapeutic polypeptide operably linkedto a promoter is an expression cassette that may include other elementsin addition to the gene and promoter, e.g. poly-adenylation sequence,enhancer elements, other genes, linker domains, etc.

By “consisting essentially of”, it is meant a limitation of the scope ofthe, for example, composition, method, kit, etc., described to thespecified materials or steps that do not materially affect the basic andnovel characteristic(s) of the, for example, composition, method, kit,etc. For example, an expression cassette “consisting essentially of” agene encoding a therapeutic polypeptide operably linked to a promoterand a polyadenylation sequence may include additional sequences, e.g.linker sequences, so long as they do not materially affect thetranscription or translation of the gene. As another example, a variant,or mutant, polypeptide fragment “consisting essentially of” a recitedsequence has the amino acid sequence of the recited sequence plus orminus about 10 amino acid residues at the boundaries of the sequencebased upon the full length naïve polypeptide from which it was derived,e.g. 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residue less than the recitedbounding amino acid residue, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10residues more than the recited bounding amino acid residue.

By “consisting of”, it is meant the exclusion from the composition,method, or kit of any element, step, or ingredient not specified in theclaim. For example, an expression cassette “consisting of” a geneencoding a therapeutic polypeptide operably linked to a promoter, and apost-transcriptional regulatory element consists only of the promoter,polynucleotide sequence encoding the therapeutic polypeptide, andpost-transcriptional regulatory element. As another example, apolypeptide “consisting of” a recited sequence contains only the recitedsequence.

An “expression vector” as used herein encompasses a vector, e.g.plasmid, minicircle, viral vector, liposome, and the like as discussedabove or as known in the art, comprising a polynucleotide encoding agene product of interest, and effecting the expression of a gene productin an intended target cell. An expression vector also comprises controlelements operatively linked to the encoding region to facilitateexpression of the gene product in the target. The combination of controlelements, e.g. promoters, enhancers, UTRs, miRNA targeting sequences,etc., and a gene or genes to which they are operably linked forexpression is sometimes referred to as an “expression cassette.” Manysuch control elements are known and available in the art or can bereadily constructed from components that are available in the art.

A “promoter” as used herein encompasses a DNA sequence that directs thebinding of RNA polymerase and thereby promotes RNA synthesis, i.e., aminimal sequence sufficient to direct transcription. Promoters andcorresponding protein or polypeptide expression may be ubiquitous,meaning strongly active in a wide range of cells, tissues and species orcell-type specific, tissue-specific, or species specific. Promoters maybe “constitutive,” meaning continually active, or “inducible,” meaningthe promoter can be activated or deactivated by the presence or absenceof biotic or abiotic factors. Also included in the nucleic acidconstructs or vectors of the invention are enhancer sequences that mayor may not be contiguous with the promoter sequence. Enhancer sequencesinfluence promoter-dependent gene expression and may be located in the5′ or 3′ regions of the native gene.

An “enhancer” as used herein encompasses a cis-acting element thatstimulates or inhibits transcription of adjacent genes. An enhancer thatinhibits transcription also is termed a “silencer”. Enhancers canfunction (i.e., can be associated with a coding sequence) in eitherorientation, over distances of up to several kilobase pairs (kb) fromthe coding sequence and from a position downstream of a transcribedregion.

A “termination signal sequence” as used herein encompasses any geneticelement that causes RNA polymerase to terminate transcription, such asfor example a polyadenylation signal sequence.

As used herein, the terms “operatively linked” or “operably linked”refers to a juxtaposition of genetic elements, e.g. promoter, enhancer,termination signal sequence, polyadenylation sequence, etc., wherein theelements are in a relationship permitting them to operate in theexpected manner. For instance, a promoter is operatively linked to acoding region if the promoter helps initiate transcription of the codingsequence. There may be intervening residues between the promoter andcoding region so long as this functional relationship is maintained.

As used herein, the term “heterologous” means derived from agenotypically distinct entity from that of the rest of the entity towhich it is being compared. For example, a polynucleotide introduced bygenetic engineering techniques into a plasmid or vector derived from adifferent species is a heterologous polynucleotide. As another example,a promoter removed from its native coding sequence and operativelylinked to a coding sequence with which it is not naturally linked is aheterologous promoter. Thus, for example, an LV vector that includes aheterologous nucleic acid encoding a heterologous gene product is an LVvector that includes a nucleic acid not normally included in anaturally-occurring, wild-type LV, and the encoded heterologous geneproduct is a gene product not normally encoded by a naturally-occurring,wild-type LV.

The term “endogenous” as used herein with reference to a nucleotidemolecule or gene product refers to a nucleic acid sequence, e.g. gene orgenetic element, or gene product, e.g. RNA, protein, that is naturallyoccurring in or associated with a host virus or cell.

The term “native” as used herein refers to a nucleotide sequence, e.g.gene, or gene product, e.g. RNA, protein, that is present in a wild-typevirus or cell.

The term “variant” as used herein refers to a mutant of a referencepolynucleotide or polypeptide sequence, for example a nativepolynucleotide or polypeptide sequence, i.e. having less than 100%sequence identity with the reference polynucleotide or polypeptidesequence. Put another way, a variant of a polynucleotide sequencecomprises at least one nucleotide difference (e.g., nucleotidesubstitution, nucleotide insertion, or nucleotide deletion) relative toa reference polynucleotide sequence, e.g., a native polynucleotidesequence. A variant of a polypeptide sequence comprises at least oneamino acid difference (e.g., amino acid substitution, amino acidinsertion, amino acid deletion) relative to a reference polypeptidesequence, e.g., a native polypeptide sequence. For example, a variantmay be a polynucleotide having a sequence identity of 70% or more with afull-length native polynucleotide sequence, e.g. an identity of 75% or80% or more, such as 85%, 90%, or 95% or more, for example, 98% or 99%identity with the full-length native polynucleotide sequence. As anotherexample, a variant may be a polypeptide having a sequence identity of70% or more with a full-length native polypeptide sequence, e.g. anidentity of 75% or 80% or more, such as 85%, 90%, or 95% or more, forexample, 98% or 99% identity with the full-length native polypeptidesequence. Variants may also include variant fragments of a reference,e.g. native, sequence sharing a sequence identity of 70% or more with afragment of the reference, e.g. native, sequence, e.g. an identity of75% or 80% or more, such as 85%, 90%, or 95% or more, for example, 98%or 99% identity with the native sequence.

As used herein, the terms “biological activity” and “biologicallyactive” refer to the activity attributed to a particular biologicalelement in a cell. For example, the “biological activity” of an“immunoglobulin”, “antibody” or fragment or variant thereof refers tothe ability to bind an antigenic determinant and thereby facilitateimmunological function. As another example, the biological activity of apolypeptide or functional fragment or variant thereof refers to theability of the polypeptide or functional fragment or variant thereof tocarry out its native functions of, e.g., binding, enzymatic activity,etc. As a third example, the biological activity of a gene regulatoryelement, e.g. promoter, enhancer, Kozak sequence, and the like, refersto the ability of the regulatory element or functional fragment orvariant thereof to regulate, i.e. promote, enhance, or activate thetranslation of, respectively, the expression of the gene to which it isoperably linked.

The terms “administering” or “introducing”, as used herein, refer todelivery of a vector for recombinant protein expression to a cell, tocells and/or organs of a subject, or to a subject. Such administering orintroducing may take place in vivo, in vitro or ex vivo. A vector forexpression of a gene product may be introduced into a cell bytransfection, which typically means insertion of heterologous DNA into acell by physical means (e.g., calcium phosphate transfection,electroporation, microinjection or lipofection); infection, whichtypically refers to introduction by way of an infectious agent, i.e., avirus; or transduction, which typically means stable infection of a cellwith a virus or the transfer of genetic material from one microorganismto another by way of a viral agent (e.g., a bacteriophage).

“Transformation” is typically used to refer to bacteria comprisingheterologous DNA, or, cells that express an oncogene and have beenconverted into a continuous growth mode such as tumor cells. A vectorused to “transform” a cell may be a plasmid, virus or other vehicle.

Typically, a cell is referred to as “transduced”, “infected”;“transfected” or “transformed” dependent on the means used foradministration, introduction or insertion of heterologous DNA (i.e., thevector) into the cell. The terms “transduced”, “transfected” and“transformed” may be used interchangeably herein regardless of themethod of introduction of heterologous DNA.

The term “host cell”, as used herein refers to a cell, which has beentransduced, infected, transfected or transformed with a vector. Thevector may be a plasmid, a viral particle, a phage, etc. The cultureconditions, such as temperature, pH and the like, are those previouslyused with the host cell selected for expression, and will be apparent tothose skilled in the art. It will be appreciated that the term “hostcell” refers to the original transduced, infected, transfected ortransformed cell and progeny thereof.

The terms “treatment”, “treating” and the like are used herein togenerally mean obtaining a desired pharmacologic and/or physiologiceffect. The effect may be prophylactic in terms of completely orpartially preventing a disease or symptom thereof, e.g. reducing thelikelihood that the disease or symptom thereof occurs in the subject,and/or may be therapeutic in terms of a partial or complete cure for adisease and/or adverse effect attributable to the disease. “Treatment”as used herein covers any treatment of a disease in a mammal, andincludes: (a) preventing the disease from occurring in a subject whichmay be predisposed to the disease but has not yet been diagnosed ashaving it; (b) inhibiting the disease, i.e., arresting its development;or (c) relieving the disease, i.e., causing regression of the disease.The therapeutic agent may be administered before, during or after theonset of disease or injury. The treatment of ongoing disease, where thetreatment stabilizes or reduces the undesirable clinical symptoms of thepatient, is of particular interest. Such treatment is desirablyperformed prior to complete loss of function in the affected tissues.The subject therapy will desirably be administered during thesymptomatic stage of the disease, and in some cases after thesymptomatic stage of the disease.

The terms “individual,” “host,” “subject,” and “patient” are usedinterchangeably herein, and refer to a mammal, including, but notlimited to, human and non-human primates, including simians and humans;mammalian sport animals (e.g., horses); mammalian farm animals (e.g.,sheep, goats, etc.); mammalian pets (dogs, cats, etc.); and rodents(e.g., mice, rats, etc.).

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention. Asused herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. Furthermore, to the extent that the terms “including”,“includes”, “having”, “has”, “with”, or variants thereof are used ineither the detailed description and/or the claims, such terms areintended to be inclusive in a manner similar to the term “comprising”.

The term “about” or “approximately” means within an acceptable errorrange for the particular value as determined by one of ordinary skill inthe art, which will depend in part on how the value is measured ordetermined, i.e., the limitations of the measurement system. Forexample, “about” can mean within 1 or more than 1 standard deviation,per the practice in the art. Alternatively, “about” can mean a range ofup to 20%, preferably up to 10%, more preferably up to 5%, and morepreferably still up to 1% of a given value. Alternatively, particularlywith respect to biological systems or processes, the term can meanwithin an order of magnitude, preferably within 5-fold, and morepreferably within 2-fold, of a value. Where particular values aredescribed in the application and claims, unless otherwise stated theterm “about” meaning within an acceptable error range for the particularvalue should be assumed.

Unless otherwise indicated, all terms used herein have the same meaningas they would to one skilled in the art and the practice of the presentinvention will employ conventional techniques of microbiology andrecombinant DNA technology, which are within the knowledge of those ofskill of the art.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of cell biology, molecular biology(including recombinant techniques), microbiology, biochemistry andimmunology, which are within the scope of those of skill in the art.Such techniques are explained fully in the literature, such as,“Molecular Cloning: A Laboratory Manual”, second edition (Sambrook etal., 1989); “Oligonucleotide Synthesis” (M. J. Gait, ed., 1984); “AnimalCell Culture” (R. I. Freshney, ed., 1987); “Methods in Enzymology”(Academic Press, Inc.); “Handbook of Experimental Immunology” (D. M.Weir & C. C. Blackwell, eds.); “Gene Transfer Vectors for MammalianCells” (J. M. Miller & M. P. Calos, eds., 1987); “Current Protocols inMolecular Biology” (F. M. Ausubel et al., eds., 1987); “PCR: ThePolymerase Chain Reaction”, (Mullis et al., eds., 1994); and “CurrentProtocols in Immunology” (J. E. Coligan et al., eds., 1991), each ofwhich is expressly incorporated by reference herein.

In certain embodiments, the present disclosure provides polynucleotides,polynucleotide cassettes and expression vectors for the expression of agene in cells. Also provided are pharmaceutical compositions and methodsfor the use of any of the compositions in promoting the expression of agene in cells, for example, in an individual, e.g. for the treatment orprophylaxis of a disorder. These and other objects, advantages, andfeatures of the invention will become apparent to those persons skilledin the art upon reading the details of the compositions and methods asmore fully described below.

The present invention relates generally to the fields of molecularbiology and virology, and in particular, to genetic expressioncassettes, and vectors comprising them useful for the delivery ofnucleic acid segments encoding selected therapeutic constructs(including for example, peptides, polypeptides, ribozymes, and catalyticRNA molecules), to selected cells and tissues of vertebrate animals. Inparticular, these genetic constructs are useful in the development ofgene therapy vectors, including for example, LV vectors, for thetreatment of mammalian, and in particular, human, diseases, disorders,and dysfunctions.

The disclosed compositions may be utilized in a variety ofinvestigative, diagnostic and therapeutic regimens, including theprevention and treatment of a variety of human diseases. The variouscompositions and methods of the invention are described below.

Although particular compositions and methods are exemplified herein, itis understood that any of a number of alternative compositions andmethods are applicable and suitable for use in practicing the invention.It will also be understood that an evaluation of the expressionconstructs and methods of the invention may be carried out usingprocedures standard in the art.

In certain embodiments, methods and compositions are provided forpreparation of gene therapy vector compositions, e.g., viral vectors,comprising these genetic expression cassettes for use in the preparationof medicaments useful in central and targeted gene therapy of diseases,disorders, and dysfunctions in an animal, and in humans in particular.

In some embodiments, the present invention provides for gene therapy forPKD based on a LV vector harbouring the PGK eukaryotic promoter thatdrives the expression of the PKLR cDNA. This therapeutic vector may beused to transduce hematopoietic stem cells (HSCs), which may besubsequently transplanted into a subject. Ectopic RPK expressionnormalizes the erythroid compartment correcting the hematologicalphenotype and reverting organ pathology. Metabolomic studies demonstratefunctional correction of the glycolytic pathway in RBCs derived fromgenetically corrected PKD HSCs, with no metabolic disturbances inleukocytes. The analysis of the LV ISs in the genome of transplantedhematopoietic cells demonstrates no evidence of genotoxicity in any ofthe transplanted animals. Overall, the results underscore thetherapeutic potential of the PGK-coRPK LV vector and provide highexpectations towards the gene therapy of PKD and other erythroidmetabolic genetic disorders.

In certain embodiments, the present invention provides an RPK LV vectorfor the genetic correction of PKD. Genetic modification of murinePKD-HSCs with this vector can efficiently correct the hemolyticphenotype and the RBC metabolite profile in transplanted PKD mice. Noevidence of metabolic disturbances in leukocytes and genotoxicityderived from the vector integration are observed, supporting thetherapeutic potential of the PGK-coRPK LV vector. Overall, resultsprovide encouraging evidence of the feasibility of gene therapy for PKDwith a LV designed for clinical application.

Certain embodiments of the present invention comprise aself-inactivating (SIN) LV vector expressing a codon-optimized versionof human PLKR gene. The expression vector comprises a promoter region, acoding sequence, and a post-transcriptional regulatory element.

Certain embodiments of polynucleotide cassettes of the present inventioncomprise a promoter region comprising a promoter sequence, or afunctional fragment thereof. In one embodiment, the promoter is a humanPGK promoter.

Some embodiments of the present invention comprise polynucleotidecassettes for the enhanced expression of pyruvate kinase (e.g., humanPLKR gene). In some embodiments, the polynucleotide cassette comprises acodon-optimized version of the human PKLR cDNA (coRPK) to increase mRNAstability upon transcription. For the optimization, GeneArt® softwaremay be used, increasing the GC content and removing cryptic splice sitesin order to avoid transcriptional silencing and therefore increasetransgene expression. The coRPK optimized sequence showed 80.4% homologywith the human PKLR gene, with no changes in the amino acids sequence ofthe protein. Alternatively, any optimization method known in the art maybe used.

In some embodiments, the polynucleotide cassette comprises an RNA exportsignal downstream of the second enhancer. The RNA export signal maycomprise a wPRE sequence. In some embodiments, a mutated wPRE, lackingany residual open reading frame (Schambach, Bohne et al. 2006) is alsoincluded to improve the level of expression and stability of thetherapeutic gene. The term “RNA export signal” may refer to any of the“post-transcriptional regulatory elements” known in the art. The terms“RNA export signal” and “post-transcriptional regulatory element” andthe like are used interchangeably herein, as they are generally in thefield of vector technology and development. Exemplary RNA export signalsor post-transcriptional regulatory elements used in some embodiments ofthe present disclosure include without limitation, hepatitis B virusposttranscriptional regulatory element (HBVPRE) or a constitutivetransport element (CTE) originating from different simian retroviruses,including simian retrovirus type 1 (SRV-1) and type-2 (SRV-2) andMason-Pfizer (MPV). Post-transcriptional regulatory elements areprovided by U.S. Pat. No. 6,136,597, Donello et al. J. Virol. 72:5085-92(1998); Hlavaty et al. Virology 341:1-11 (2005); and Oh et al.Retrovirology 4:38 (2007). Further alternative wPRE sequences areprovided by Zanta-Boussif et al. Gene Therapy 16:605-19 (2009). Thedisclosures of the foregoing reference are incorporated herein in theirentirety.

In some aspects of the invention, gene delivery vectors are providedcomprising a polynucleotide cassette of the present invention. In someembodiments, the gene delivery vector is a LV.

In some aspects of the invention, pharmaceutical compositions areprovided comprising a polynucleotide cassette of the invention and apharmaceutical excipient. In some embodiments, the pharmaceuticalcomposition comprises a gene delivery vector of the invention and apharmaceutical excipient.

In some aspects of the invention, methods are provided for expressing atransgene in mammalian cells. In some embodiments, the method comprisescontacting one or more mammalian cells with an effective amount of apolynucleotide cassette of the invention or a gene delivery vector ofthe invention, wherein the transgene is expressed at detectable levelsin the one or more mammalian cells. In some embodiments, the methodcomprises contacting one or more mammalian cells with an effectiveamount of a polynucleotide cassette of the invention or a gene deliveryvector of the invention, wherein the transgene is expressed attherapeutic levels in the one or more mammalian cells. In someembodiments, the method is in vitro. In other embodiments, the method isin vivo.

In some aspects of the invention, methods are provided for the treatmentor prophylaxis of a disease or disorder in a mammal in need of treatmentor prophylaxis for a disease or disorder. In some embodiments, themethod comprises administering to the mammal an effective amount of apharmaceutical composition of the invention, wherein the coding sequenceencodes a therapeutic gene product.

Compositions

In some aspects of the disclosure, compositions are provided for theexpression of a transgene in a eukaryotic cell(s). In some aspects, theeukaryotic cell is a mammalian cell. In some aspects, the mammalian cellis a hematopoietic stem cell. In some embodiments, the cell is a bonemarrow cell, e.g., a lineage depleted bone marrow cell. In some aspects,the mammalian cell is a committed hematopoietic erythroid progenitor.

In some embodiments of the disclosure, the composition is apolynucleotide cassette. By a “polynucleotide cassette” is meant apolynucleotide sequence comprising two or more functional polynucleotidesequences, e.g. regulatory elements, translation initiation sequences,coding sequences, and/or termination sequences, etc., typically inoperable linkage to one another. Likewise, by a “polynucleotide cassettefor the expression of a transgene in a mammalian cell,” it is meant acombination of two or more functional polynucleotide sequences, e.g.promoter, enhancer, 5′UTR, translation initiation sequence, codingsequence, and/or termination sequences, etc. that promotes theexpression of the transgene in a cell.

In some embodiments, the polynucleotide cassettes of the presentdisclosure provide for enhanced expression of a transgene in mammaliancells. As used herein “expression of a transgene” or “expressing atransgene” refers to both the transcription of the transgene into amessenger RNA (mRNA) by the host cell and translation of that mRNA intoa polypeptide (i.e., the transgene product). Thus, expression of thetransgene can be measured either at the mRNA level (i.e., bysequence-specific quantification of the level of mRNA in a cell) or atthe polypeptide level (i.e., by measuring the level of polypeptide geneproduct using a Western Blot, Enzyme-Linked Immunoabsorbent Assay(ELISA), Immunofluorescence Microscopy, or other means of quantifying aspecific polypeptide). As demonstrated by the working examples of thepresent disclosure, the present inventors have discovered a number ofpolynucleotide elements, i.e., improved elements as compared to thoseknown in the art, which individually and synergistically provide for theenhanced expression of transgenes in mammalian cells. In certainembodiments, the arrangement of the two or more functionalpolynucleotide sequences within the polynucleotide cassettes of thepresent disclosure provide for enhanced expression of a transgene inmammalian cells. By “enhanced” it is meant that expression of thetransgene is increased, augmented, or stronger, in cells carrying thepolynucleotide cassettes of the present disclosure relative to in cellscarrying the transgene operably linked to comparable regulatoryelements, e.g. as known in the art. Put another way, expression of thetransgene is increased, augmented, or stronger, from the polynucleotidecassettes of the present disclosure relative to expression from apolynucleotide cassette not comprising the one or more optimizedelements of the present disclosure, i.e. a reference control. In certainembodiments, the enhanced expression is specific for or limited to oneor more desired cell types.

For example, expression of the transgene may be enhanced, or augmented,or increased, in cells comprising a polynucleotide cassette comprising apromoter disclosed herein than in cells that carry the transgeneoperably linked to a different promoter, e.g. as known in the art. Asanother example, expression of the transgene may be enhanced, orincreased, augmented, or stronger, in cells comprising a polynucleotidecassette comprising an enhancer sequence disclosed herein than in cellsthat carry the transgene operably linked to a different enhancersequence.

Without wishing to be bound by theory, enhanced expression of atransgene in cells is believed to be due to a more rapid build-up ofgene product in the cells or a more stable gene product in the cells.Thus, enhanced expression of a transgene by the polynucleotide cassettesof the subject disclosure may be observed in a number of ways. Forexample, enhanced expression may be observed by detecting the expressionof the transgene following contact of the polynucleotide cassette to thecells sooner, e.g. 2 days sooner, 7 days sooner, 2 weeks sooner, 3 weekssooner, 4 weeks sooner, 8 weeks sooner, 12 weeks sooner or more, thanexpression would be detected if the transgene were operably linked tocomparable regulatory elements, e.g., as known in the art. Enhancedexpression may also be observed as an increase in the amount of geneproduct per cell. For example, there may be a 2-fold increase or more,e.g. a 3-fold increase or more, a 4-fold increase or more, a 5-foldincrease or more, or a 10-fold increase or more in the amount of geneproduct per mammalian cell. Enhanced expression may also be observed asan increase in the number of mammalian cells that express detectablelevels of the transgene carried by the polynucleotide cassette. Forexample, there may be a 2-fold increase or more, e.g. a 3-fold increaseor more, a 4-fold increase or more, a 5-fold increase or more, or a10-fold increase or more in the number of mammalian cells that expressdetectable levels of the transgene.

As another example, the polynucleotide of the present invention maypromote detectable levels of the transgene in a greater percentage ofcells as compared to a conventional polynucleotide cassette; forexample, where a conventional cassette may promote detectable levels oftransgene expression in, for example, less than 5% of the cells in acertain region, the polynucleotide of the present invention promotesdetectable levels of expression in 5% or more of the cells in thatregion; e.g. 10% or more, 15% or more, 20% or more, 25% or more, 30% ormore, 35% or more, 40% or more, or 45% or more, in some instances 50% ormore, 55% or more; 60% or more, 65% or more, 70% or more, or 75% ormore, for example 80% or more, 85% or more, 90% or more, or 95% or moreof the cells that are contacted, will express detectable levels of geneproduct. Enhanced expression may also be observed as an alteration inthe viability and/or function of the cells.

The polynucleotide cassettes of the present disclosure typicallycomprise a promoter region. Any suitable promoter region or promotersequence therein can be used in the subject polynucleotide cassettes, solong as the promoter region promotes expression of a coding sequence ineukaryotic cells. In certain embodiments, the promoter region promotesexpression of a coding sequence in mammalian cells. In some instances,the promoter is a ubiquitous promoter, i.e., it is a promoter that isactive in a wide range of cells, tissues and species. In otherinstances, the promoter is a human PGK promoter.

Promoter and enhancer elements can be tissue specific or stage-specific.For example, a tissue-specific promoter or enhancer preferentiallydrives expression (or a higher level of expression) in one or moreparticular cell types. Examples of cell types include but are notlimited to: hematopoietic stem cells, long term hematopoietic stemcells, short term hematopoietic stem cells, multipotent progenitors,hematopoietic CD34+ cells and any cluster of differentiationsubpopulation within the CD34+ population. A stage-specific promoter orenhancer preferentially drives expression (or higher level ofexpression) during one or more specific stages of the cell cycle ordevelopment. These include but are not limited to beta-globin locuscontrol region, spectrin promoter, and an erythroid specific promoter.

In some embodiments, the polynucleotide comprises one or more enhancers.Enhancers are nucleic acid elements known in the art to enhancetranscription, and can be located anywhere in association with the genethey regulate, e.g. upstream, downstream, within an intron, etc. Anyenhancer element can be used in the polynucleotide cassettes and genetherapy vectors of the present disclosure, so long as it enhancesexpression of the gene when used in combination with the promoter.

The coding sequence to be expressed in the cells can be anypolynucleotide sequence, e.g. gene or cDNA that encodes a gene product,e.g. a polypeptide or RNA-based therapeutic (siRNA, antisense, ribozyme,shRNA, etc.). The coding sequence may be heterologous to the promotersequence to which it is operably linked, i.e. not naturally operablyassociated with it. Alternatively, the coding sequence may be endogenousto the promoter sequence to which it is operably linked, i.e. isassociated in nature with that promoter. The gene product may actintrinsically in the mammalian cell, or it may act extrinsically, e.g.,it may be secreted. For example, when the transgene is a therapeuticgene, the coding sequence may be any gene that encodes a desired geneproduct or functional fragment or variant thereof that can be used as atherapeutic for treating a disease or disorder. In various preferredembodiments, the transgene encodes human PKLR.

In one embodiment of the invention, the transgene coding sequence ismodified, or “codon optimized” to enhance expression by replacinginfrequently represented codons with more frequently represented codons.The coding sequence is the portion of the mRNA sequence that encodes theamino acids for translation. During translation, each of 61trinucleotide codons are translated to one of 20 amino acids, leading toa degeneracy, or redundancy, in the genetic code. However, differentcell types, and different animal species, utilize tRNAs (each bearing ananticodon) coding for the same amino acids at different frequencies.When a gene sequence contains codons that are infrequently representedby the corresponding tRNA, the ribosome translation machinery may slow,impeding efficient translation. Expression can be improved via “codonoptimization” for a particular species, where the coding sequence isaltered to encode the same protein sequence, but utilizing codons thatare highly represented, and/or utilized by highly expressed humanproteins (Cid-Arregui et al., 2003; J. Virol. 77: 4928). In one aspectof the present invention, the coding sequence of the transgene ismodified to replace codons infrequently expressed in mammal or inprimates with codons frequently expressed in primates. For example, insome embodiments, the coding sequence encoded by the transgene encodes apolypeptide having at least 85% sequence identity to a polypeptideencoded by a sequence disclosed above or herein, for example at least90% sequence identity, e.g. at least 95% sequence identity, at least 98%identity, at least 99% identity, wherein at least one codon of thecoding sequence has a higher tRNA frequency in humans than thecorresponding codon in the sequence disclosed above or herein.

In an additional embodiment of the invention, the transgene codingsequence is modified to enhance expression by termination or removal ofopen reading frames (ORFs) that do not encode the desired transgene. Anopen reading frame (ORF) is the nucleic acid sequence that follows astart codon and does not contain a stop codon. ORFs may be in theforward or reverse orientation, and may be “in frame” or “out of frame”compared with the gene of interest. Such open reading frames have thepotential to be expressed in an expression cassette alongside the geneof interest, and could lead to undesired adverse effects. In one aspectof the present invention, the coding sequence of the transgene has beenmodified to remove open reading frames by further altering codon usage.This was done by eliminating start codons (ATG) and introducing stopcodons (TAG, TAA, or TGA) in reverse orientation or out-of-frame ORFs,while preserving the amino acid sequence and maintaining highly utilizedcodons in the gene of interest (i.e., avoiding codons with frequency<20%). In the present invention, the transgene coding sequence may beoptimized by either of codon optimization and removal of non-transgeneORFs or using both techniques. As will be apparent to one of ordinaryskill in the art, it is preferable to remove or minimize non-transgeneORFs after codon optimization in order to remove ORFs introduced duringcodon optimization.

The RRE sequence improves the efficiency of gene transfer. In particularembodiments of any of the expression cassettes and gene delivery vectorsdescribed herein, the RRE sequence comprises or consists of thefollowing sequence, or a sequence having at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, or at least 99% identity to thefollowing sequence:

(SEQ ID NO: 1) AGGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTGACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACAGCTCCT.

The retroviral leader region contains the packaging signal (Ψ), which isinvolved in packaging the retroviral genome into the viral capsid. LVvectors were thought to require approximately 300 bp of the Gag gene inthis region. Currently, this Gag sequence has been reduced to just 40 bp(FIG. 1 ). In particular embodiments of any of the expression cassettesand gene delivery vectors described herein, the w sequence is an HIV-1 wsequence or the w sequence comprises or consists of the followingsequence, or a sequence having at least 80%, at least 85%, at least 90%,at least 95%, at least 98%, or at least 99% identity to the followingsequence:

(SEQ ID NO: 2) CTCTCTCGACGCAGGACTCGGCTTGCTGAAGCGCGCACGGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACGCCAAAAATTTTGACTAGCGGAGGCTAGAAGGAGAGAGATGGGTGCGAGAGCGTC.

In particular embodiments of any of the expression cassettes and genedelivery vectors described herein, the truncated HIV-1 5′ LTR comprisesor consists of the following sequence, or a sequence having at least80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least99% identity to the following sequence:

(SEQ ID NO: 3) GGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCA.

In particular embodiments of any of the expression cassettes and genedelivery vectors described herein, the HIV-1 self-inactivating 3′ LTRcomprises or consists of the following sequence, or a sequence having atleast 80%, at least 85%, at least 90%, at least 95%, at least 98%, or atleast 99% identity to the following sequence:

(SEQ ID NO: 4) TGGAAGGGCTAATTCACTCCCAACGAAGACAAGATCTGCTTTTTGCTTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCA.

In particular embodiments of any of the expression cassettes and genedelivery vectors described herein, the human cytomegalovirus (CMV)immediate early promoter comprises or consists of the followingsequence, a functional fragment thereof, or a sequence having at least80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least99% identity to the following sequence:

(SEQ ID NO: 5) GTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAG AGCT.

The cPPT, which facilitates nuclear translocation of the pre-integrationcomplexes, together with the CTS involved in the separation of reversetranscriptase, has been seen to improve viral titer (Zennou, et al.2000; Follenzi et al. 2000). In particular embodiments of any of theexpression cassettes and gene delivery vectors described herein, thecentral polypurine tract and central termination sequence of HIV-1(cPPT/CTS) comprises or consists of the following sequence, a functionalfragment thereof, or a sequence having at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, or at least 99% identity to thefollowing sequence:

(SEQ ID NO: 6) TTTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTGCAGGGGAAAGAATAGTAGACATAATAGCAACAGACATACAAACTAAAGAATTACAAAAACAAATT ACAAAAATTCAAAATTTT.

In particular embodiments of any of the expression cassettes and genedelivery vectors described herein, the human phosphoglycerate kinase 1(hPGK) promoter comprises or consists of the following sequence, afunctional fragment thereof, or a sequence having at least 80%, at least85%, at least 90%, at least 95%, at least 98%, or at least 99% identityto the following sequence:

(SEQ ID NO: 7) GGGGTTGGGGTTGCGCCTTTTCCAAGGCAGCCCTGGGTTTGCGCAGGGACGCGGCTGCTCTGGGCGTGGTTCCGGGAAACGCAGCGGCGCCGACCCTGGGTCTCGCACATTCTTCACGTCCGTTCGCAGCGTCACCCGGATCTTCGCCGCTACCCTTGTGGGCCCCCCGGCGACGCTTCCTGCTCCGCCCCTAAGTCGGGAAGGTTCCTTGCGGTTCGCGGCGTGCCGGACGTGACAAACGGAAGCCGCACGTCTCACTAGTACCCTCGCAGACGGACAGCGCCAGGGAGCAATGGCAGCGCGCCGACCGCGATGGGCTGTGGCCAATAGCGGCTGCTCAGCAGGGCGCGCCGAGAGCAGCGGCCGGGAAGGGGCGGTGCGGGAGGCGGGGTGTGGGGCGGTAGTGTGGGCCCTGTTCCTGCCCGCGCGGTGTTCCGCATTCTGCAAGCCTCCGGAGCGCACGTCGGCAGTCGGCTCCCTCGTTGACCGAATCACCGACC TCTCTCCCCAG.

In particular embodiments of any of the expression cassettes and genedelivery vectors described herein, the codon-optimized version of thehuman PKLR cDNA (coRPK) comprises or consists of the following sequence,a functional fragment thereof, or a sequence having at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, or at least 99%identity to the following sequence:

(SEQ ID NO: 8) ATGAGCATCCAGGAAAATATCAGCTCTCTGCAGCTGCGGTCCTGGGTGTCCAAGAGCCAGAGAGACCTGGCCAAGAGCATCCTGATCGGAGCCCCTGGCGGACCAGCCGGATACCTGAGAAGGGCTAGCGTGGCCCAGCTGACCCAGGAACTGGGCACCGCCTTTTTCCAGCAGCAGCAGCTGCCAGCCGCCATGGCCGACACCTTTCTGGAACACCTGTGCCTGCTGGACATCGACTCTGAGCCCGTGGCCGCCAGAAGCACCAGCATCATTGCCACCATCGGCCCTGCCAGCAGAAGCGTGGAGCGGCTGAAAGAGATGATCAAGGCCGGCATGAATATCGCCCGGCTGAACTTCTCCCACGGCAGCCACGAGTACCACGCAGAGAGCATTGCCAACGTCCGGGAGGCCGTGGAGAGCTTTGCCGGCAGCCCCCTGAGCTACAGACCCGTGGCCATTGCCCTGGACACCAAGGGCCCCGAGATCAGAACAGGAATTCTGCAGGGAGGGCCTGAGAGCGAGGTGGAGCTGGTGAAGGGCAGCCAAGTGCTGGTGACCGTGGACCCCGCCTTCAGAACCAGAGGCAACGCCAACACAGTGTGGGTGGACTACCCCAACATCGTGCGGGTGGTGCCTGTGGGCGGCAGAATCTACATCGACGACGGCCTGATCAGCCTGGTGGTGCAGAAGATCGGACCTGAGGGCCTGGTGACCCAGGTCGAGAATGGCGGCGTGCTGGGCAGCAGAAAGGGCGTGAATCTGCCAGGCGCCCAGGTGGACCTGCCTGGCCTGTCTGAGCAGGACGTGAGAGACCTGAGATTTGGCGTGGAGCACGGCGTGGACATCGTGTTCGCCAGCTTCGTGCGGAAGGCCTCTGATGTGGCCGCCGTGAGAGCCGCTCTGGGCCCTGAAGGCCACGGCATCAAGATCATCAGCAAGATCGAGAACCACGAGGGCGTGAAGCGGTTCGACGAGATCCTGGAAGTGTCCGACGGCATCATGGTGGCCAGAGGCGACCTGGGCATCGAGATCCCCGCCGAGAAGGTGTTCCTGGCCCAGAAAATGATGATCGGACGGTGCAACCTGGCCGGCAAACCTGTGGTGTGCGCCACCCAGATGCTGGAAAGCATGATCACCAAGCCCAGACCCACCAGAGCCGAGACAAGCGACGTGGCCAACGCCGTGCTGGATGGCGCTGACTGCATCATGCTGTCCGGCGAGACAGCCAAGGGCAACTTCCCCGTGGAGGCCGTGAAGATGCAGCACGCCATTGCCAGAGAAGCCGAGGCCGCCGTGTACCACCGGCAGCTGTTCGAGGAACTGCGGAGAGCCGCCCCTCTGAGCAGAGATCCCACCGAAGTGACCGCCATCGGAGCCGTGGAAGCCGCCTTCAAGTGCTGCGCCGCTGCAATCATCGTGCTGACCACCACAGGCAGAAGCGCCCAGCTGCTGTCCAGATACAGACCCAGAGCCGCCGTGATCGCCGTGACAAGATCCGCCCAGGCCGCTAGACAGGTCCACCTGTGCAGAGGCGTGTTCCCCCTGCTGTACCGGGAGCCTCCCGAGGCCATCTGGGCCGACGACGTGGACAGACGGGTGCAGTTCGGCATCGAGAGCGGCAAGCTGCGGGGCTTCCTGAGAGTGGGCGACCTGGTGATCGTGGTGACAGGCTGGCGGCCTGGCAGCGGCTACACCAACATCATGAGGGTGCTGTCCATCAGCTGA.

In particular embodiments of any of the expression cassettes and genedelivery vectors described herein, the human CMV enhancer comprises orconsists of the following sequence, a functional fragment thereof, or asequence having at least 80%, at least 85%, at least 90%, at least 95%,at least 98%, or at least 99% identity to the following sequence:

(SEQ ID NO: 9) GACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATG.

In particular embodiments of any of the expression cassettes and genedelivery vectors described herein, the simian virus 40 (SV40) poly(A)signal comprises or consists of the following sequence, a functionalfragment thereof, or a sequence having at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, or at least 99% identity to thefollowing sequence:

(SEQ ID NO: 10) AACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTA.

In particular embodiments of any of the expression cassettes and genedelivery vectors described herein, the SV40 origin of replicationcomprises or consists of the following sequence, a functional fragmentthereof, or a sequence having at least 80%, at least 85%, at least 90%,at least 95%, at least 98%, or at least 99% identity to the followingsequence:

(SEQ ID NO: 11) ATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCC.

In particular embodiments of any of the expression cassettes and genedelivery vectors described herein, the cPPT present in any of theexpression cassettes or gene delivery vectors described herein comprisesor consists of the following sequence: TTTAAAAGAAAAGGGGGGATTGGGGGGT (SEQID NO:12), or a sequence having at least 80%, at least 85%, at least90%, at least 95%, at least 98%, or at least 99% identity to SEQ IDNO:12.

In some embodiments of any of the expression cassettes and gene deliveryvectors described herein, the dNEF signal present in any of theexpression cassettes or gene delivery vectors described herein comprisesor consists of the following sequence:GAATTCGAGCTCGGTACCTTTAAGACCAATGACTTACAAGGCAGCTGTAGATCTTAGCCACTTTTTAAAAGAAAAGGGGGGAC (SEQ ID NO:13), or a sequence having atleast 80%, at least 85%, at least 90%, at least 95%, at least 98%, or atleast 99% identity to SEQ ID NO:13.

In particular embodiments of any of the expression cassettes and genedelivery vectors described herein, the NeoR/KanR sequence present in anyof the expression cassettes or gene delivery vectors described hereincomprises or consists of the following sequence:ATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCGGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGTCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAAGACGAGGCAGCGCGGCTATCGTGGCTGGCGACGACGGGCGTTCCTTGCGCGGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGTCTATGCCCGACGGCGAGGATCTCGTCGTGACCCACGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGTCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTTGTGCTTTACGGTATCGCCGCGCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGA (SEQ ID NO:14), or a sequence havingat least 80%, at least 85%, at least 90%, at least 95%, at least 98%, orat least 99% identity to SEQ ID NO:14.

In some embodiments of any of the expression cassettes and gene deliveryvectors described herein, the rrnG terminator (transcription terminatorfrom the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991))present in any of the expression cassettes or gene delivery vectorsdescribed herein comprises or consists of the following sequence:GCATTGGCGCAGAAAAAAATGCCTGATGCGACGCTGCGCGTCTTATACTCCCACATATGCCAGATTCAGCAACGGATACGGCTTCCCCAACTTGCCCACTTCCATACGTGTCCTCCTTACCAGAAATTTATCCTTAA (SEQ ID NO:15), or a sequence having atleast 80%, at least 85%, at least 90%, at least 95%, at least 98%, or atleast 99% identity to SEQ ID NO:15.

In some embodiments of any of the expression cassettes and gene deliveryvectors described herein, the ori (high-copy-numberColE1/pMB1/pBR322/pUC origin of replication) present in any of theexpression cassettes or gene delivery vectors described herein comprisesor consists of the following sequence:TTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAA (SEQ ID NO: 16), or asequence having at least 80%, at least 85%, at least 90%, at least 95%,at least 98%, or at least 99% identity to SEQ ID NO:16.

In some embodiments of any of the expression cassettes and gene deliveryvectors described herein, the CAP binding site present in any of theexpression cassettes or gene delivery vectors described herein comprisesor consists of the following sequence: TAATGTGAGTTAGCTCACTCAT (SEQ IDNO:17), or a sequence having at least 80%, at least 85%, at least 90%,at least 95%, at least 98%, or at least 99% identity to SEQ ID NO:17.

In some embodiments of any of the expression cassettes and gene deliveryvectors described herein, the E. coli lac promoter present in any of theexpression cassettes or gene delivery vectors described herein comprisesor consists of the following sequence: TTTACACTTTATGCTTCCGGCTCGTATGTTG(SEQ ID NO:18), or a sequence having at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, or at least 99% identity to SEQID NO:18.

In some embodiments of any of the expression cassettes and gene deliveryvectors described herein, the lac operator present in any of theexpression cassettes or gene delivery vectors described herein comprisesor consists of the following sequence: TTGTGAGCGGATAACAA (SEQ ID NO:19), or a sequence having at least 80%, at least 85%, at least 90%, atleast 95%, at least 98%, or at least 99% identity to SEQ ID NO: 19.

In some embodiments of any of the expression cassettes and gene deliveryvectors described herein, the T3 promoter (promoter for bacteriophage T3RNA polymerase) present in any of the expression cassettes or genedelivery vectors described herein comprises or consists of the followingsequence: AATTAACCCTCACTAAAGG (SEQ ID NO: 20), or a sequence having atleast 80%, at least 85%, at least 90%, at least 95%, at least 98%, or atleast 99% identity to SEQ ID NO: 20.

In some embodiments of any of the expression cassettes and gene deliveryvectors described herein, the CMV enhancer present in any of theexpression cassettes or gene delivery vectors described herein comprisesor consists of the following sequence:GACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATG (SEQ ID NO: 21), or asequence having at least 80%, at least 85%, at least 90%, at least 95%,at least 98%, or at least 99% identity to SEQ ID NO: 21.

In some embodiments of any of the expression cassettes and gene deliveryvectors described herein, the T7 promoter (promoter for bacteriophage T7RNA polymerase) present in any of the expression cassettes or genedelivery vectors described herein comprises or consists of the followingsequence: CCTATAGTGAGTCGTATTA (SEQ ID NO: 22), or a sequence having atleast 80%, at least 85%, at least 90%, at least 95%, at least 98%, or atleast 99% identity to SEQ ID NO: 22.

In some embodiments of any of the expression cassettes and gene deliveryvectors described herein, the fl ori (fl bacteriophage origin ofreplication) present in any of the expression cassettes or gene deliveryvectors described herein comprises or consists of the followingsequence: ACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATT (SEQ ID NO: 23), or asequence having at least 80%, at least 85%, at least 90%, at least 95%,at least 98%, or at least 99% identity to SEQ ID NO: 23.

In some embodiments, the polynucleotide cassette of the presentinvention further comprises an RNA export signal. Exemplary RNA exportsequences include but are not limited to wPRE. The wPRE significantlyincreases transgene expression in target cells, by increasing RNAstability in a transgene, promoter and vector-independent manner(Zuffrey et al, 1999). However, it can express a truncated 60-amino acidprotein derived from the WHV X gene involved in liver cancer (Kingsmanet al, 2005). Therefore, most pre-clinical protocols and clinical trialsinclude a mutated version of the wPRE element (Zanta-Boussif et al,2009). On the other hand, the use of two SV40-USE elements in SIN-LVvectors has been seen to be more efficient than the wPRE sequence insuppressing transcriptional read through (Schambach et al, 2007). Moreprecisely, the wPRE disclosed herein is a chimeric wPRE that carries 589nucleotides from the modified WPRE performed by Axel Schambach(nucleotides 1-589) (WO 2008136670 A2; [5]) and 88 from a former wPRE(nucleotide 590-677) (Zuffrey et al, 1999). Data disclosed herein showsthis chimeric wPRE works better than the former wPRE. The chimeric wPREsequence comprises the following sequence:

(SEQ ID NO: 24) CGAGCATCTTACCGCCATTTATTCCCATATTTGTTCTGTTTTTCTTGATTTGGGTATACATTTAAATGTTAATAAAACAAAATGGTGGGGCAATCATTTACATTTTTAGGGATATGTAATTACTAGTTCAGGTGTATTGCCACAAGACAAACATGTTAAGAAACTTTCCCGTTATTTACGCTCTGTTCCTGTTAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGATATTCTTAACTATGTTGCTCCTTTTACGCTGTGTGGATATGCTGCTTTAATGCCTCTGTATCATGCTATTGCTTCCCGTACGGCTTTCGTTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCCGTCAACGTGGCGTGGTGTGCTCTGTGTTTGCTGACGCAACCCCCACTGGCTGGGGCATTGCCACCACCTGTCAACTCCTTTCTGGGACTTTCGCTTTCCCCCTCCCGATCGCCACGGCAGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTAGGTTGCTGGGCACTGATAATTCCGTGGTGTTGTCGGGGAAGGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCCTG.

In particular embodiments of any of the expression cassettes and genedelivery vectors described herein, the wPRE sequence comprises orconsists of the sequence of SEQ ID NO:24, or a sequence having at least80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least99% identity to the sequence of SEQ ID NO:24.

(SEQ ID NO: 24) CGAGCATCTTACCGCCATTTATTCCCATATTTGTTCTGTTTTTCTTGATTTGGGTATACATTTAAATGTTAATAAAACAAAATGGTGGGGCAATCATTTACATTTTTAGGGATATGTAATTACTAGTTCAGGTGTATTGCCACAAGACAAACATGTTAAGAAACTTTCCCGTTATTTACGCTCTGTTCCTGTTAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGATATTCTTAACTATGTTGCTCCTTTTACGCTGTGTGGATATGCTGCTTTAATGCCTCTGTATCATGCTATTGCTTCCCGTACGGCTTTCGTTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCCGTCAACGTGGCGTGGTGTGCTCTGTGTTTGCTGACGCAACCCCCACTGGCTGGGGCATTGCCACCACCTGTCAACTCCTTTCTGGGACTTTCGCTTTCCCCCTCCCGATCGCCACGGCAGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTAGGTTGCTGGGCACTGATAATTCCGTGGTGTTGTCGGGGAAGGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCCTG

Other combinations of elements both as disclosed herein or as known inthe art will be readily appreciated by the ordinarily skilled artisan.

Additionally, as will be recognized by one of ordinary skill in the art,the polynucleotide cassettes may optionally contain other elementsincluding, but not limited to restriction sites to facilitate cloningand regulatory elements for a particular gene expression vector.

In some aspects of the present invention, the subject polynucleotidecassettes are used to deliver a gene to cells of an animal, e.g. todetermine the effect that the gene has on cell viability and/orfunction, to treat a cell disorder, etc. Accordingly, in some aspects ofthe invention, the composition that provides for the expression of atransgene in mammalian cells is a gene delivery vector, wherein the genedelivery vector comprises the polynucleotide cassettes of the presentdisclosure.

Any convenient gene therapy vector that finds use deliveringpolynucleotide sequences to mammalian cells is encompassed by the genedelivery vectors of the present disclosure. For example, the vector maycomprise single or double stranded nucleic acid, e.g. single stranded ordouble stranded DNA. For example, the gene delivery vector may be DNA,e.g., a naked DNA, e.g., a plasmid, a minicircle, etc. The vector maycomprise single-stranded or double-stranded RNA, including modifiedforms of RNA. In another example, the gene delivery vector may be anRNA, e.g., an mRNA or modified mRNA.

As another example, the gene delivery vector may be a viral vectorderived from a virus, e.g., an adenovirus, an adeno-associated virus, aLV, a herpes virus, an alphavirus or a retrovirus, e.g., Moloney murineleukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), Harveymurine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV),gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV),spumavirus, Friend murine leukemia virus, Murine Stem Cell Virus (MSCV)or Rous Sarcoma Virus (RSV). While embodiments encompassing the use ofLV are described in greater detail below, it is expected that theordinarily skilled artisan will appreciate that similar knowledge andskill in the art can be brought to bear on non-LV gene therapy vectorsas well.

In some embodiments, the gene delivery vector is a self-limiting LV. Insome embodiments, the gene delivery vector is a self-inactivating LV. Insome embodiments, the gene delivery vector is a non-integrating LV. In aspecific embodiment of any of the expression cassettes and gene deliveryvectors described herein, the self-limiting LV (FIGS. 22 and 23 ) of thedisclosure comprises or consists of the following sequence, or asequence having at least 80%, at least 85%, at least 90%, at least 95%,at least 98%, or at least 99% identity to the following sequence:

(SEQ ID NO: 25) GTGTTTAAACCTAGATATTGATAGTCTGATCGGTCAACGTATAATCGAGTCCTAGCTTTTGCAAACATCTATCAAGAGACAGGATCAGCAGGAGGCTTTCGCATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCGGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGTCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAAGACGAGGCAGCGCGGCTATCGTGGCTGGCGACGACGGGCGTTCCTTGCGCGGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGTCTATGCCCGACGGCGAGGATCTCGTCGTGACCCACGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGTCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTTGTGCTTTACGGTATCGCCGCGCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGACCGATTCTAGGTGCATTGGCGCAGAAAAAAATGCCTGATGCGACGCTGCGCGTCTTATACTCCCACATATGCCAGATTCAGCAACGGATACGGCTTCCCCAACTTGCCCACTTCCATACGTGTCCTCCTTACCAGAAATTTATCCTTAACGATCGGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCGCGCAATTAACCCTCACTAAAGGGAACAAAAGCTGGAGCTGCAAGCTTGGCCATTGCATACGTTGTATCCATATCATAATATGTACATTTATATTGGCTCATGTCCAACATTACCGCCATGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTGGCGCCCGAACAGGGACTTGAAAGCGAAAGGGAAACCAGAGGAGCTCTCTCGACGCAGGACTCGGCTTGCTGAAGCGCGCACGGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACGCCAAAAATTTTGACTAGCGGAGGCTAGAAGGAGAGAGATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGATCGCGATGGGAAAAAATTCGGTTAAGGCCAGGGGGAAAGAAAAAATATAAATTAAAACATATAGTATGGGCAAGCAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAACATCAGAAGGCTGTAGACAAATACTGGGACAGCTACAACCATCCCTTCAGACAGGATCAGAAGAACTTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTGCATCAAAGGATAGAGATAAAAGACACCAAGGAAGCTTTAGACAAGATAGAGGAAGAGCAAAACAAAAGTAAGACCACCGCACAGCAAGCGGCCGCTGATCTTCAGACCTGGAGGAGGAGATATGAGGGACAATTGGAGAAGTGAATTATATAAATATAAAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACCAAGGCAAAGAGAAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTGACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACAGCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACTGCTGTGCCTTGGAATGCTAGTTGGAGTAATAAATCTCTGGAACAGATTTGGAATCACACGACCTGGATGGAGTGGGACAGAGAAATTAACAATTACACAAGCTTAATACACTCCTTAATTGAAGAATCGCAAAACCAGCAAGAAAAGAATGAACAAGAATTATTGGAATTAGATAAATGGGCAAGTTTGTGGAATTGGTTTAACATAACAAATTGGCTGTGGTATATAAAATTATTCATAATGATAGTAGGAGGCTTGGTAGGTTTAAGAATAGTTTTTGCTGTACTTTCTATAGTGAATAGAGTTAGGCAGGGATATTCACCATTATCGTTTCAGACCCACCTCCCAACCCCGAGGGGACCCGACAGGCCCGAAGGAATAGAAGAAGAAGGTGGAGAGAGAGACAGAGACAGATCCATTCGATTAGTGAACGGATCTCGACGGTATCGGTTAACTTTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTGCAGGGGAAAGAATAGTAGACATAATAGCAACAGACATACAAACTAAAGAATTACAAAAACAAATTACAAAAATTCAAAATTTTATCGATCACGAGACTAGCCTCGAGAAGCTTGATATCGAATTCCACGGGGTTGGGGTTGCGCCTTTTCCAAGGCAGCCCTGGGTTTGCGCAGGGACGCGGCTGCTCTGGGCGTGGTTCCGGGAAACGCAGCGGCGCCGACCCTGGGTCTCGCACATTCTTCACGTCCGTTCGCAGCGTCACCCGGATCTTCGCCGCTACCCTTGTGGGCCCCCCGGCGACGCTTCCTGCTCCGCCCCTAAGTCGGGAAGGTTCCTTGCGGTTCGCGGCGTGCCGGACGTGACAAACGGAAGCCGCACGTCTCACTAGTACCCTCGCAGACGGACAGCGCCAGGGAGCAATGGCAGCGCGCCGACCGCGATGGGCTGTGGCCAATAGCGGCTGCTCAGCAGGGCGCGCCGAGAGCAGCGGCCGGGAAGGGGCGGTGCGGGAGGCGGGGTGTGGGGCGGTAGTGTGGGCCCTGTTCCTGCCCGCGCGGTGTTCCGCATTCTGCAAGCCTCCGGAGCGCACGTCGGCAGTCGGCTCCCTCGTTGACCGAATCACCGACCTCTCTCCCCAGGGGGATCCGTCGACACCGGTGCCACCATGAGCATCCAGGAAAATATCAGCTCTCTGCAGCTGCGGTCCTGGGTGTCCAAGAGCCAGAGAGACCTGGCCAAGAGCATCCTGATCGGAGCCCCTGGCGGACCAGCCGGATACCTGAGAAGGGCTAGCGTGGCCCAGCTGACCCAGGAACTGGGCACCGCCTTTTTCCAGCAGCAGCAGCTGCCAGCCGCCATGGCCGACACCTTTCTGGAACACCTGTGCCTGCTGGACATCGACTCTGAGCCCGTGGCCGCCAGAAGCACCAGCATCATTGCCACCATCGGCCCTGCCAGCAGAAGCGTGGAGCGGCTGAAAGAGATGATCAAGGCCGGCATGAATATCGCCCGGCTGAACTTCTCCCACGGCAGCCACGAGTACCACGCAGAGAGCATTGCCAACGTCCGGGAGGCCGTGGAGAGCTTTGCCGGCAGCCCCCTGAGCTACAGACCCGTGGCCATTGCCCTGGACACCAAGGGCCCCGAGATCAGAACAGGAATTCTGCAGGGAGGGCCTGAGAGCGAGGTGGAGCTGGTGAAGGGCAGCCAAGTGCTGGTGACCGTGGACCCCGCCTTCAGAACCAGAGGCAACGCCAACACAGTGTGGGTGGACTACCCCAACATCGTGCGGGTGGTGCCTGTGGGCGGCAGAATCTACATCGACGACGGCCTGATCAGCCTGGTGGTGCAGAAGATCGGACCTGAGGGCCTGGTGACCCAGGTCGAGAATGGCGGCGTGCTGGGCAGCAGAAAGGGCGTGAATCTGCCAGGCGCCCAGGTGGACCTGCCTGGCCTGTCTGAGCAGGACGTGAGAGACCTGAGATTTGGCGTGGAGCACGGCGTGGACATCGTGTTCGCCAGCTTCGTGCGGAAGGCCTCTGATGTGGCCGCCGTGAGAGCCGCTCTGGGCCCTGAAGGCCACGGCATCAAGATCATCAGCAAGATCGAGAACCACGAGGGCGTGAAGCGGTTCGACGAGATCCTGGAAGTGTCCGACGGCATCATGGTGGCCAGAGGCGACCTGGGCATCGAGATCCCCGCCGAGAAGGTGTTCCTGGCCCAGAAAATGATGATCGGACGGTGCAACCTGGCCGGCAAACCTGTGGTGTGCGCCACCCAGATGCTGGAAAGCATGATCACCAAGCCCAGACCCACCAGAGCCGAGACAAGCGACGTGGCCAACGCCGTGCTGGATGGCGCTGACTGCATCATGCTGTCCGGCGAGACAGCCAAGGGCAACTTCCCCGTGGAGGCCGTGAAGATGCAGCACGCCATTGCCAGAGAAGCCGAGGCCGCCGTGTACCACCGGCAGCTGTTCGAGGAACTGCGGAGAGCCGCCCCTCTGAGCAGAGATCCCACCGAAGTGACCGCCATCGGAGCCGTGGAAGCCGCCTTCAAGTGCTGCGCCGCTGCAATCATCGTGCTGACCACCACAGGCAGAAGCGCCCAGCTGCTGTCCAGATACAGACCCAGAGCCGCCGTGATCGCCGTGACAAGATCCGCCCAGGCCGCTAGACAGGTCCACCTGTGCAGAGGCGTGTTCCCCCTGCTGTACCGGGAGCCTCCCGAGGCCATCTGGGCCGACGACGTGGACAGACGGGTGCAGTTCGGCATCGAGAGCGGCAAGCTGCGGGGCTTCCTGAGAGTGGGCGACCTGGTGATCGTGGTGACAGGCTGGCGGCCTGGCAGCGGCTACACCAACATCATGAGGGTGCTGTCCATCAGCTGACCGCGGTCTAGAGGATCCCCCGGGCTGCAGGAATTCGAGCATCTTACCGCCATTTATTCCCATATTTGTTCTGTTTTTCTTGATTTGGGTATACATTTAAATGTTAATAAAACAAAATGGTGGGGCAATCATTTACATTTTTAGGGATATGTAATTACTAGTTCAGGTGTATTGCCACAAGACAAACATGTTAAGAAACTTTCCCGTTATTTACGCTCTGTTCCTGTTAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGATATTCTTAACTATGTTGCTCCTTTTACGCTGTGTGGATATGCTGCTTTAATGCCTCTGTATCATGCTATTGCTTCCCGTACGGCTTTCGTTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCCGTCAACGTGGCGTGGTGTGCTCTGTGTTTGCTGACGCAACCCCCACTGGCTGGGGCATTGCCACCACCTGTCAACTCCTTTCTGGGACTTTCGCTTTCCCCCTCCCGATCGCCACGGCAGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTAGGTTGCTGGGCACTGATAATTCCGTGGTGTTGTCGGGGAAGGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCCTGGAATTCGAGCTCGGTACCTTTAAGACCAATGACTTACAAGGCAGCTGTAGATCTTAGCCACTTTTTAAAAGAAAAGGGGGGACTGGAAGGGCTAATTCACTCCCAACGAAGACAAGATCTGCTTTTTGCTTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTAGTAGTTCATGTCATCTTATTATTCAGTATTTATAACTTGCAAAGAAATGAATATCAGAGAGTGAGAGGAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGCTCTAGCTATCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGGACGTACCCAATTCGCCCTATAGTGAGTCGTATTACGCGCGCTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATCGTTCCCTCAGGACGTC.

In a specific embodiment of the expression cassettes described herein,the expression comprises or consists of the following sequence, or asequence having at least 80%, at least 85%, at least 90%, at least 95%,at least 98%, or at least 99% identity to the following sequence:

(SEQ ID NO: 26) GGGGTTGGGGTTGCGCCTTTTCCAAGGCAGCCCTGGGTTTGCGCAGGGACGCGGCTGCTCTGGGCGTGGTTCCGGGAAACGCAGCGGCGCCGACCCTGGGTCTCGCACATTCTTCACGTCCGTTCGCAGCGTCACCCGGATCTTCGCCGCTACCCTTGTGGGCCCCCCGGCGACGCTTCCTGCTCCGCCCCTAAGTCGGGAAGGTTCCTTGCGGTTCGCGGCGTGCCGGACGTGACAAACGGAAGCCGCACGTCTCACTAGTACCCTCGCAGACGGACAGCGCCAGGGAGCAATGGCAGCGCGCCGACCGCGATGGGCTGTGGCCAATAGCGGCTGCTCAGCAGGGCGCGCCGAGAGCAGCGGCCGGGAAGGGGCGGTGCGGGAGGCGGGGTGTGGGGCGGTAGTGTGGGCCCTGTTCCTGCCCGCGCGGTGTTCCGCATTCTGCAAGCCTCCGGAGCGCACGTCGGCAGTCGGCTCCCTCGTTGACCGAATCACCGACCTCTCTCCCCAGGGGGATCCGTCGACACCGGTGCCACCATGAGCATCCAGGAAAATATCAGCTCTCTGCAGCTGCGGTCCTGGGTGTCCAAGAGCCAGAGAGACCTGGCCAAGAGCATCCTGATCGGAGCCCCTGGCGGACCAGCCGGATACCTGAGAAGGGCTAGCGTGGCCCAGCTGACCCAGGAACTGGGCACCGCCTTTTTCCAGCAGCAGCAGCTGCCAGCCGCCATGGCCGACACCTTTCTGGAACACCTGTGCCTGCTGGACATCGACTCTGAGCCCGTGGCCGCCAGAAGCACCAGCATCATTGCCACCATCGGCCCTGCCAGCAGAAGCGTGGAGCGGCTGAAAGAGATGATCAAGGCCGGCATGAATATCGCCCGGCTGAACTTCTCCCACGGCAGCCACGAGTACCACGCAGAGAGCATTGCCAACGTCCGGGAGGCCGTGGAGAGCTTTGCCGGCAGCCCCCTGAGCTACAGACCCGTGGCCATTGCCCTGGACACCAAGGGCCCCGAGATCAGAACAGGAATTCTGCAGGGAGGGCCTGAGAGCGAGGTGGAGCTGGTGAAGGGCAGCCAAGTGCTGGTGACCGTGGACCCCGCCTTCAGAACCAGAGGCAACGCCAACACAGTGTGGGTGGACTACCCCAACATCGTGCGGGTGGTGCCTGTGGGCGGCAGAATCTACATCGACGACGGCCTGATCAGCCTGGTGGTGCAGAAGATCGGACCTGAGGGCCTGGTGACCCAGGTCGAGAATGGCGGCGTGCTGGGCAGCAGAAAGGGCGTGAATCTGCCAGGCGCCCAGGTGGACCTGCCTGGCCTGTCTGAGCAGGACGTGAGAGACCTGAGATTTGGCGTGGAGCACGGCGTGGACATCGTGTTCGCCAGCTTCGTGCGGAAGGCCTCTGATGTGGCCGCCGTGAGAGCCGCTCTGGGCCCTGAAGGCCACGGCATCAAGATCATCAGCAAGATCGAGAACCACGAGGGCGTGAAGCGGTTCGACGAGATCCTGGAAGTGTCCGACGGCATCATGGTGGCCAGAGGCGACCTGGGCATCGAGATCCCCGCCGAGAAGGTGTTCCTGGCCCAGAAAATGATGATCGGACGGTGCAACCTGGCCGGCAAACCTGTGGTGTGCGCCACCCAGATGCTGGAAAGCATGATCACCAAGCCCAGACCCACCAGAGCCGAGACAAGCGACGTGGCCAACGCCGTGCTGGATGGCGCTGACTGCATCATGCTGTCCGGCGAGACAGCCAAGGGCAACTTCCCCGTGGAGGCCGTGAAGATGCAGCACGCCATTGCCAGAGAAGCCGAGGCCGCCGTGTACCACCGGCAGCTGTTCGAGGAACTGCGGAGAGCCGCCCCTCTGAGCAGAGATCCCACCGAAGTGACCGCCATCGGAGCCGTGGAAGCCGCCTTCAAGTGCTGCGCCGCTGCAATCATCGTGCTGACCACCACAGGCAGAAGCGCCCAGCTGCTGTCCAGATACAGACCCAGAGCCGCCGTGATCGCCGTGACAAGATCCGCCCAGGCCGCTAGACAGGTCCACCTGTGCAGAGGCGTGTTCCCCCTGCTGTACCGGGAGCCTCCCGAGGCCATCTGGGCCGACGACGTGGACAGACGGGTGCAGTTCGGCATCGAGAGCGGCAAGCTGCGGGGCTTCCTGAGAGTGGGCGACCTGGTGATCGTGGTGACAGGCTGGCGGCCTGGCAGCGGCTACACCAACATCATGAGGGTGCTGTCCATCAGCTGACCGCGGTCTAGAGGATCCCCCGGGCTGCAGGAATTCGAGCATCTTACCGCCATTTATTCCCATATTTGTTCTGTTTTTCTTGATTTGGGTATACATTTAAATGTTAATAAAACAAAATGGTGGGGCAATCATTTACATTTTTAGGGATATGTAATTACTAGTTCAGGTGTATTGCCACAAGACAAACATGTTAAGAAACTTTCCCGTTATTTACGCTCTGTTCCTGTTAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGATATTCTTAACTATGTTGCTCCTTTTACGCTGTGTGGATATGCTGCTTTAATGCCTCTGTATCATGCTATTGCTTCCCGTACGGCTTTCGTTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCCGTCAACGTGGCGTGGTGTGCTCTGTGTTTGCTGACGCAACCCCCACTGGCTGGGGCATTGCCACCACCTGTCAACTCCTTTCTGGGACTTTCGCTTTCCCCCTCCCGATCGCCACGGCAGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTAGGTTGCTGGGCACTGATAATTCCGTGGTGTTGTCGGG GAAGGGCC.

In such embodiments, the subject polynucleotide cassette is flanked onthe 5′ and 3′ ends by functional LTR sequences. In one embodiment, theposition of different elements present in the backbone of the LV vectoris depicted in FIG. 1 . Both LTR sequences have been modified togenerate SIN LV vectors. SIN vectors have a 400 bp deletion in the3′-LTR, covering the promoter/enhancer elements from the U3 region.Expression of the transgene is thereby dependent on internal promoters,reducing the risk of RCLs and decreasing promoter interference (Ginn etal, 2003). This 3′-LTR deletion removes the TATA box, preventingtranscription initiation (Miyoshi et al. 1998; Zuffrey et al 1998); andtherefore inactivating the vector. The U3 region of the 5′-LTR has beenreplaced by other heterologous promoting sequences (i.e. CMV or RSV) toachieve a Tat-independent transcription and to increase genomic RNAsynthesis, resulting in the increase of the viral titer. Because 5′-U3region drives the expression of primary transcripts, its modificationswill not be present in transduced cells (Schambach et al. 2009).

Exogenous elements, such as β-globin or SV40 polyadenylation signals(Iwakuma et al, 1999) or the USE from SV40-USE (Schambach et al. 2007),have also been included in the R region of the viral 3′LTR in order todecrease the transcriptional readthrough from the internal promoters(Zaiss et al, 2002) or from remnants of the deleted U3 region of SIN-LVvectors (Almarza et al. 2011) preventing the potential transcriptionalactivation of the downstream genes.

In certain embodiments, an expression cassette or gene delivery vector,e.g., a LV, comprises a polynucleotide sequence comprising the followingsequences in 5′ to 3′ order:

-   -   a) a PGK promoter sequence, optionally a human PGK promoter        sequence;    -   b) a sequence encoding a pyruvate kinase polypeptide, optionally        a coRPK coding or cDNA sequence; and    -   c) a mutant wPRE sequence, optionally comprising or consisting        of the sequence of SEQ ID NO:24.

In certain embodiments, an expression cassette or gene delivery vector,e.g., a LV, comprises a polynucleotide sequence comprising the followingsequences in 5′ to 3′ order:

-   -   a) a cPPT sequence;    -   b) PGK promoter sequence, optionally a human PGK promoter        sequence;    -   c) a sequence encoding a pyruvate kinase polypeptide, optionally        a coRPK coding or cDNA sequence; and    -   d) a mutant wPRE sequence, optionally comprising or consisting        of the sequence of SEQ ID NO:24.

In certain embodiments, an expression cassette or gene delivery vector,e.g., a LV, comprises a polynucleotide sequence comprising the followingsequences in 5′ to 3′ order:

-   -   a) a 5′ LTR, optionally a modified 5′ LTR;    -   b) a cPPT sequence;    -   c) PGK promoter sequence, optionally a human PGK promoter        sequence;    -   d) a sequence encoding a pyruvate kinase polypeptide, optionally        a coRPK coding or cDNA sequence;    -   e) a mutant wPRE sequence, optionally comprising or consisting        of the sequence of SEQ ID NO:24; and    -   f) a 3′ LTR, optionally a modified 3′ LTR.

In certain embodiments, an expression cassette or gene delivery vector,e.g., a LV, comprises a polynucleotide sequence comprising the followingsequences in 5′ to 3′ order:

-   -   a) a 5′ LTR, optionally a modified 5′ LTR;    -   b) a cPPT sequence;    -   c) PGK promoter sequence, optionally a human PGK promoter        sequence;    -   d) a sequence encoding a pyruvate kinase polypeptide, optionally        a coRPK coding or cDNA sequence;    -   e) a mutant wPRE sequence, optionally comprising or consisting        of the sequence of SEQ ID NO:24;    -   f) a 3′ LTR, optionally a modified 3′ LTR;    -   g) an SV40 poly(A) signal; and    -   h) an SV40 origin of replication (ori).

In certain embodiments, an expression cassette or gene delivery vector,e.g., a LV, comprises a polynucleotide sequence comprising the followingsequences in 5′ to 3′ order:

-   -   a) a 5′ LTR, optionally a modified 5′ LTR;    -   b) a cPPT sequence;    -   c) PGK promoter sequence, optionally a human PGK promoter        sequence;    -   d) a sequence encoding a pyruvate kinase polypeptide, optionally        a coRPK coding or cDNA sequence;    -   e) a mutant wPRE sequence, optionally comprising or consisting        of the sequence of SEQ ID NO:24;    -   f) a 3′ LTR, optionally a modified 3′ LTR;    -   g) an SV40 poly(A) signal;    -   h) an SV40 origin of replication (ori);    -   i) a T7 promoter;    -   j) an fl ori; and    -   k) a NeoR/KanR sequence.

In certain embodiments, an expression cassette or gene delivery vector,e.g., a LV, comprises a polynucleotide sequence comprising the followingsequences in 5′ to 3′ order:

-   -   a) a 5′ LTR, optionally a modified 5′ LTR;    -   b) an HIV-1 w sequence;    -   c) an RRE;    -   d) a cPPT/CTS sequence;    -   e) a PGK promoter sequence, optionally a human PGK promoter        sequence;    -   f) a sequence encoding a pyruvate kinase polypeptide, optionally        a coRPK coding or cDNA sequence;    -   g) a mutant wPRE sequence, optionally comprising or consisting        of the sequence of SEQ ID NO:24;    -   h) a dNEF signal;    -   i) a 3′ LTR, optionally a modified 3′ LTR;    -   j) an SV40 poly(A) signal;    -   k) an SV40 origin of replication (ori);    -   l) a T7 promoter;    -   m) an fl ori;    -   n) a NeoR/KanR sequence;    -   o) a rmG terminator;    -   p) an ori sequence;    -   q) a CAP binding site;    -   r) a lac promoter sequence;    -   s) a lac operator sequence;    -   t) a T3 promoter sequence;    -   u) a CMV enhancer sequence; and    -   v) a CMV promoter sequence.

In certain embodiments, the gene delivery vector is PGK-coRPK LV, orcomprises the elements depicted in FIG. 21 .

In certain embodiments, an expression cassette or gene delivery vector,e.g., a LV, comprises a polynucleotide sequence comprising the followingsequences in 5′ to 3′ order:

-   -   a) a 5′ LTR, optionally a modified 5′ LTR;    -   b) an HIV-1 w sequence;    -   c) an RRE;    -   d) a cPPT/CTS sequence;    -   e) a PGK promoter sequence, optionally a human PGK promoter        sequence;    -   f) a sequence encoding a pyruvate kinase polypeptide, optionally        a coRPK coding or cDNA sequence;    -   g) a mutant wPRE sequence, optionally comprising or consisting        of the sequence of SEQ ID NO: 24;    -   h) optionally, a dNEF signal;    -   i) a 3′ LTR, optionally a modified 3′ LTR;    -   j) an SV40 poly(A) signal;    -   k) an SV40 origin of replication (ori);    -   1) a T7 promoter;    -   m) pUC ori;    -   u) a CMV enhancer sequence; and    -   v) a CMV promoter sequence.

In particular embodiments of any of the expression cassettes and genedelivery vectors described herein, the coRPK cDNA or coding sequenceencodes a PKLR polypeptide that comprises or consists of the sequencedisclosed in any of GenBank accession Nos. XP_016856982.1,XP_011507942.1, XP_006711449.1, NP_870986.1, or NP_000289.1, or afunctional fragment of any of these sequences, or a sequence having atleast 80%, at least 85%, at least 90%, at least 95%, at least 98%, or atleast 99% identity to any of these sequences.

Gene therapy vectors encapsulating the polynucleotide cassettes of thepresent disclosure may be produced using standard methodology. Forexample, in the case of LV virions, an LV expression vector according tothe invention may be introduced into a producer cell, followed byintroduction of an LV helper construct, where the helper constructincludes LV coding regions capable of being expressed in the producercell and which complement LV helper functions absent in the LV vector.This is followed by introduction of helper virus and/or additionalvectors into the producer cell, wherein the helper virus and/oradditional vectors provide accessory functions capable of supportingefficient LV virus production. The producer cells are then cultured toproduce LV. These steps are carried out using standard methodology.

Any suitable method for producing viral particles for delivery of thesubject polynucleotide cassettes can be used, including but not limitedto those described in the examples that follow. Any concentration ofviral particles suitable to effectively transducer mammalian cells canbe prepared for contacting mammalian cells in vitro or in vivo. Forexample, the viral particles may be formulated at a concentration of 10⁸vector genomes per ml or more, for example, 5×10⁸ vector genomes per mL;10⁹ vector genomes per mL; 5×10⁹ vector genomes per mL, 10¹⁰ vectorgenomes per mL, 5×10¹⁰ vector genomes per mL; 10¹¹ vector genomes permL; 5×10¹¹ vector genomes per mL; 10¹² vector genomes per mL; 5×10¹²vector genomes per mL; 10¹³ vector genomes per mL; 1.5×10¹³ vectorgenomes per mL; 3×10¹³ vector genomes per mL; 5×10¹³ vector genomes permL; 7.5×10¹³ vector genomes per mL; 9×10¹³ vector genomes per mL; 1×10¹⁴vector genomes per mL, 5×10¹⁴ vector genomes per mL or more, buttypically not more than 1×10¹⁵ vector genomes per mL.

In preparing the subject LV compositions, any host cells for producingLV virions may be employed, including, for example, mammalian cells(e.g. 293 cells), insect cells (e.g. SF9 cells), microorganisms andyeast. Host cells can also be packaging cells in which the LV rep andcap genes are stably maintained in the host cell or producer cells inwhich the LV vector genome is stably maintained and packaged. Exemplarypackaging and producer cells are derived from SF-9, 293, A549 or HeLacells. LV vectors are purified and formulated using standard techniquesknown in the art.

In certain embodiments, the present invention includes a cell comprisingan expression cassette or gene delivery vector disclosed herein. Inrelated embodiments, the cell is transduced with a viral vectorcomprising an expression cassette disclosed herein or has an expressioncassette disclosed herein integrated into the cell's genome. In certainembodiments, the cell is a cell used to produce a viral gene deliveryvector. In other embodiments, the cell is a cell to be delivered to asubject in order to provide to the subject the gene product encoded bythe expression cassette. Thus, in certain embodiments, the cell isautologous to the subject to be treated or was obtained from the subjectto be treated. In other embodiments, the cell is allogeneic to thesubject to be treated or was obtained from a donor other than thesubject to be treated. In particular embodiments, the cell is amammalian cell, e.g., a human cell. In certain embodiments, the cell isa blood cell, an erythrocyte, a hematopoietic progenitor cell, a bonemarrow cell, e.g., a lineage depleted bone marrow cell, a hematopoieticstem cell (e.g., CD34+) or a committed hematopoietic erythroidprogenitor cell.

The present invention includes pharmaceutical compositions comprising apolynucleotide cassette, gene delivery vector, or cell described hereinand a pharmaceutically-acceptable carrier, diluent or excipient. Thesubject polynucleotide cassette, gene delivery vector, or cell can becombined with pharmaceutically-acceptable carriers, diluents andreagents useful in preparing a formulation that is generally safe,non-toxic, and desirable, and includes excipients that are acceptablefor primate use. Such excipients can be solid, liquid, semisolid, or, inthe case of an aerosol composition, gaseous. Examples of suchexcipients, carriers or diluents include, but are not limited to, water,saline, Ringer's solutions, dextrose solution, and 5% human serumalbumin. Supplementary active compounds can also be incorporated intothe formulations. Solutions or suspensions used for the formulations caninclude a sterile diluent such as water for injection, saline solution,fixed oils, polyethylene glycols, glycerine, propylene glycol or othersynthetic solvents; antibacterial compounds such as benzyl alcohol ormethyl parabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating compounds such as ethylenediaminetetraacetic acid (EDTA);buffers such as acetates, citrates or phosphates; detergents such asTween 20 to prevent aggregation; and compounds for the adjustment oftonicity such as sodium chloride or dextrose. The pH can be adjustedwith acids or bases, such as hydrochloric acid or sodium hydroxide. Inparticular embodiments, the pharmaceutical compositions are sterile.

Pharmaceutical compositions suitable for use in the present inventionfurther include sterile aqueous solutions or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion.

Sterile solutions can be prepared by incorporating the active compoundin the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, methods of preparation are vacuum dryingand freeze-drying that yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof

In one embodiment, the compositions are prepared with carriers that willprotect the gene cassette or expression vector against rapid eliminationfrom the body, such as a controlled release formulation, includingimplants and microencapsulated delivery systems. Biodegradable,biocompatible polymers can be used, such as ethylene vinyl acetate,polyanhydrides, polyglycolic acid, collagen, polyorthoesters, andpolylactic acid. Methods for preparation of such formulations will beapparent to those skilled in the art. The materials can also be obtainedcommercially.

It is especially advantageous to formulate oral, ocular or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

The pharmaceutical compositions can be included in a container, pack, ordispenser, e.g. syringe, e.g. a prefilled syringe, together withinstructions for administration.

The pharmaceutical compositions of the invention encompass anypharmaceutically acceptable salts, esters, or salts of such esters, orany other compound which, upon administration to an animal comprising ahuman, is capable of providing (directly or indirectly) the biologicallyactive metabolite or residue thereof.

The term “pharmaceutically acceptable salt” refers to physiologicallyand pharmaceutically acceptable salts of the compounds of the invention:i.e., salts that retain the desired biological activity of the parentcompound and do not impart undesired toxicological effects thereto. Avariety of pharmaceutically acceptable salts are known in the art anddescribed, e.g., in in “Remington's Pharmaceutical Sciences”, 17thedition, Alfonso R. Gennaro (Ed.), Mark Publishing Company, Easton, PA,USA, 1985 (and more recent editions thereof), in the “Encyclopaedia ofPharmaceutical Technology”, 3rd edition, James Swarbrick (Ed.), InformaHealthcare USA (Inc.), NY, USA, 2007, and in J. Pharm. Sci. 66: 2(1977). Also, for a review on suitable salts, see Handbook ofPharmaceutical Salts: Properties, Selection, and Use by Stahl andWermuth (Wiley-VCH, 2002).

Pharmaceutically acceptable base addition salts are formed with metalsor amines, such as alkali and alkaline earth metals or organic amines.Metals used as cations comprise sodium, potassium, magnesium, calcium,and the like. Amines comprise N—N′-dibenzylethylenediamine,chloroprocaine, choline, diethanolamine, dicyclohexylamine,ethylenediamine, N-methylglucamine, and procaine (see, for example,Berge et al., “Pharmaceutical Salts,” J. Pharma Sci., 1977, 66, 119).The base addition salts of said acidic compounds are prepared bycontacting the free acid form with a sufficient amount of the desiredbase to produce the salt in the conventional manner. The free acid formmay be regenerated by contacting the salt form with an acid andisolating the free acid in the conventional manner. The free acid formsdiffer from their respective salt forms somewhat in certain physicalproperties such as solubility in polar solvents, but otherwise the saltsare equivalent to their respective free acid for purposes of the presentinvention.

The subject polynucleotide cassette, gene delivery vector, e.g.,recombinant virus (virions), or cell (e.g., transduced with a genedelivery vector disclosed herein) can be incorporated intopharmaceutical compositions for administration to mammalian patients,particularly primates and more particularly humans. The subjectpolynucleotide cassette, gene delivery vector, e.g. virions, or cell canbe formulated in nontoxic, inert, pharmaceutically acceptable aqueouscarriers, preferably at a pH ranging from 3 to 8, more preferablyranging from 6 to 8. Such sterile compositions will comprise the vectoror virion containing the nucleic acid encoding the therapeutic moleculedissolved in an aqueous buffer having an acceptable pH uponreconstitution.

In some embodiments, the pharmaceutical composition provided hereincomprise a therapeutically effective amount of a cell, vector or viriondisclosed herein in admixture with a pharmaceutically acceptable carrierand/or excipient, for example saline, phosphate buffered saline,phosphate and amino acids, polymers, polyols, sugar, buffers,preservatives and other proteins. Exemplary amino acids, polymers andsugars and the like are octylphenoxy polyethoxy ethanol compounds,polyethylene glycol monostearate compounds, polyoxyethylene sorbitanfatty acid esters, sucrose, fructose, dextrose, maltose, glucose,mannitol, dextran, sorbitol, inositol, galactitol, xylitol, lactose,trehalose, bovine or human serum albumin, citrate, acetate, Ringer's andHank's solutions, cysteine, arginine, carnitine, alanine, glycine,lysine, valine, leucine, polyvinylpyrrolidone, polyethylene and glycol.Preferably, this formulation is stable for at least six months at 4° C.

In some embodiments, the pharmaceutical composition provided hereincomprises a buffer, such as phosphate buffered saline (PBS) or sodiumphosphate/sodium sulfate, tris buffer, glycine buffer, sterile water andother buffers known to the ordinarily skilled artisan such as thosedescribed by Good et al. (1966) Biochemistry 5:467. The pH of the bufferin which the pharmaceutical composition comprising the tumor suppressorgene contained in the adenoviral vector delivery system, may be in therange of 6.5 to 7.75, preferably 7 to 7.5, and most preferably 7.2 to7.4.

In certain embodiments, viral vectors may be formulated into anysuitable unit dosage, including, without limitation, 1×10⁸ vectorgenomes or more, for example, 1×10⁹, 1×10¹⁰, 1×10¹¹, 1×10¹², or 1×10¹³vector genomes or more, in certain instances, 1×10¹⁴ vector genomes, butusually no more than 4×10¹⁵ vector genomes. In some cases, the unitdosage is at most about 5×10¹⁵ vector genomes, e.g. 1×10¹⁴ vectorgenomes or less, for example 1×10¹³, 1×10¹², 1×10¹¹, 1×10¹⁰, or 1×10⁹vector genomes or less, in certain instances 1×10⁸ vector genomes orless, and typically no less than 1×10⁸ vector genomes. In some cases,the unit dosage is 1×10¹⁰ to 1×10¹¹ vector genomes. In some cases, theunit dosage is 1×10¹⁰ to 3×10¹² vector genomes. In some cases, the unitdosage is 1×10⁹ to 3×10¹³ vector genomes. In some cases, the unit dosageis 1×10⁸ to 3×10¹⁴ vector genomes. In one embodiment, the range is fromabout 5×10¹⁰ to about 1×10¹¹ vector genomes. In some embodiments, therange is from about 1×10⁹ to about 1×10¹⁰ vector genomes.

In some cases, the unit dosage of a pharmaceutical composition may bemeasured using multiplicity of infection (MOI). By MOI it is meant theratio, or multiple, of vector or viral genomes to the cells to which thenucleic acid may be delivered. In some cases, the MOI may be 1×10⁶. Insome cases, the MOI may be 1×10⁵-1×10⁷. In some cases, the MOI may be1×10⁴-1×10⁸. In some cases, recombinant viruses of the disclosure are atleast about 1×10¹, 1×10², 1×10³, 1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸,1×10⁹, 1×10¹⁰, 1×10¹¹, 1×10¹², 1×10¹³, 1×10¹⁴, 1×10¹⁵, 1×10¹⁶, 1×10¹⁷,and 1×10¹⁸ MOI. In some cases, recombinant viruses of this disclosureare 1×10⁸ to 3×10¹⁴ MOI. In some cases, recombinant viruses of thedisclosure are at most about 1×10¹, 1×10², 1×10³, 1×10⁴, 1×10⁵, 1×10⁶,1×10⁷, 1×10⁸, 1×10⁹, 1×10¹⁰, 1×10¹¹, 1×10¹², 1×10¹³, 1×10¹⁴, 1×10¹⁵,1×10¹⁶, 1×10¹⁷, and 1×10¹⁸ MOI. In some, embodiments the range is fromabout 20 to about 400 MOI.

In some aspects, the amount of pharmaceutical composition comprisesabout 1×10⁸ to about 1×10¹⁵ recombinant viruses, about 1×10⁹ to about1×10¹⁴ recombinant viruses, about 1×10¹⁰ to about 1×10¹³ recombinantviruses, or about 1×10¹¹ to about 3×10¹² recombinant viruses.

Methods

As disclosed herein, the subject polynucleotide cassettes and genedelivery vectors, referred to collectively herein as the “subjectcompositions”, find use in expressing a transgene in cells of an animal.For example, the subject compositions may be used in research, e.g. todetermine the effect that the gene has on cell viability and/orfunction. As another example, the subject compositions may be used inmedicine, e.g. to treat or prevent a disease or disorder. Thus, in someaspects of the invention, methods are provided for the expression of agene in cells, the method comprising contacting cells with a compositionof the present disclosure. In some embodiments, contacting occurs invitro or ex vivo. In some embodiments, contacting occurs in vivo, i.e.,the subject composition is administered to a subject.

For instances in which mammalian cells are to be contacted in vitro orex vivo with a subject polynucleotide cassette or gene delivery vectorcomprising a subject polynucleotide cassette, the cells may be from anymammalian species, e.g. rodent (e.g. mice, rats, gerbils, squirrels),rabbit, feline, canine, goat, ovine, pig, equine, bovine, primate,human. Cells may be from established cell lines or they may be primarycells, where “primary cells”, “primary cell lines”, and “primarycultures” are used interchangeably herein to refer to cells and cellscultures that have been derived from a subject and allowed to grow invitro for a limited number of passages, i.e. splittings, of the culture.For example, primary cultures are cultures that may have been passaged 0times, 1 time, 2 times, 4 times, 5 times, 10 times, or 15 times, but notenough times go through the crisis stage. Typically, the primary celllines of the present invention are maintained for fewer than 10 passagesin vitro.

In particular embodiments, the cell to be contacted with a disclosedpolynucleotide cassette or gene delivery vector are hematopoietic stemcells (HSCs) from the subject or from a donor, e.g., an allogenic donor.In some embodiments, a biological sample, e.g., peripheral blood, isobtained from the subject following mobilization of hematopoietic stemcells (HSCs). In one embodiment, HSCs and/or progenitor cells aremobilized by treating the subject with G-CSF or an analog thereof. HSCsand progenitor cells (HSPC) in peripheral blood may be mobilized priorto collection of the biological sample. Peripheral blood HSCs and HSPCcan be mobilized by any method known in the art. Peripheral blood HSCsand HSPC can be mobilized by treating the subject with any agent(s),described herein or known in the art, that increase the number of HSPCcirculating in the peripheral blood of the subject. For example, inparticular embodiments, peripheral blood is mobilized by treating thesubject with one or more cytokines or growth factors (e.g., G-CSF, kitligand (1 L), IL-I, IL-7, IL-8, IL-11, Flt3 ligand, SCF, thrombopoietin,or GM-CSF (such as sargramostim)). Different types of G-CSF that can beused in the methods for mobilization of peripheral blood includefilgrastim and longer acting G-CSF: pegfilgrastim. In particularembodiments, peripheral blood is mobilized by treating the subject withone or more chemokines (e.g., macrophage inflammatory protein-1a(MIP1a/CCL3)), chemokine receptor ligands (e.g., chemokine receptor 2ligands GRO13 and GRO13M), chemokine receptor analogs (e.g., stromalcell derived factor-1a (SDF-1a) protein analogs such as CTCE-0021,CTCE-0214, or SDF-1a such as Met-SDF-113), or chemokine receptorantagonists (e.g., chemokine (C-X-C motif) receptor 4 (CXCR4)antagonists such as AMD3100). In particular embodiments, peripheralblood is mobilized by treating the subject with one or moreanti-integrin signaling agents (e.g., function blocking anti-very lateantigen 4 (VLA-4) antibody, or anti-vascular cell adhesion molecule 1(VCAM-1)). In particular embodiments, peripheral blood is mobilized bytreating the subject with one or more cytotoxic drugs such ascyclophosphamide, etoposide or paclitaxel. In particular embodiments,peripheral blood can be mobilized by administering to a subject one ormore of the agents listed above for a certain period of time. Forexample, the subject can be treated with one or more agents (e.g.,G-CSF) via injection (e.g., subcutaneous, intravenous orintraperitoneal), once daily or twice daily, for 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13 or 14 days prior to collection of HSPC. In specificembodiments, HSPC are collected within 1, 2, 3, 4, 5, 6, 7, 8, 12, 14,16, 18, 20 or 24 hours after the last dose of an agent used formobilization of HSPC into peripheral blood. In particular embodiments,HSCs and HSPC are mobilized by treating the subject with two or moredifferent types of agents described above or known in the art, such as agrowth factor (e.g., G-CSF) and a chemokine receptor antagonist (e.g.,CXCR4 receptor antagonist such as AMD3100), or a growth factor (e.g.,G-CSF or KL) and an anti-integrin agent (e.g., function blocking VLA-4antibody). In one embodiment, HSCs and/or progenitor cells are mobilizedby treating the subject with G-CSF or an analog thereof. In oneembodiment, the G-CSF is filgrastim. In one embodiment, HSCs and/orprogenitor cells are mobilized by treating the subject with plerixafor.In a certain embodiment, HSCs and/or progenitor cells are mobilizedusing a combination of filgrastim and plerixafor, by filgrastim alone,or by plerixafor alone. In particular embodiments, different types ofmobilizing agents are administered concurrently or sequentially. Foradditional information regarding methods of mobilization of peripheralblood see, e.g., Craddock et al., 1997, Blood 90(12):4779-4788; Jin etal., 2008, Journal of Translational Medicine 6:39; Pelus, 2008, Curr.Opin. Hematol. 15(4):285-292; Papayannopoulou et al., 1998, Blood91(7):2231-2239; Tricot et al., 2008, Haematologica 93(11):1739-1742;and Weaver et al., 2001, Bone Marrow Transplantation 27(2): S23-S29).

In some embodiments, the cells are enriched prior to contacting thecells with the disclosed polynucleotide cassette or gene deliveryvector. As used herein, “enriched” or “high enriched” refers to a methodof enriching for a cell population intended to result in substantialenrichment of cells for cells expressing a particular biological marker,e.g. CD34. For example, CD34 enrichment used clinically results in mean:61.6% and median: 65.7% yield of CD34⁺ cells and mean: 88.5% and median:95.9% relative purity (N=166) (Clin Lab. 2016 Jul. 1; 62(7):1243-1248(PMID: 28164638)). “Enrichment” refers to a process with the goal ofsubstantial enrichment of a relatively rare cell type, CD34+, whichusually comprises between 0.2-2% of the cell product in a mobilizedleukopheresis or bone marrow collection. Enrichment of CD34+ cells froma mobilized leukopheresis or bone marrow collection targets final CD34+percentages that have increased from 0.2-2% to >80%. To accomplish this,following initial application of a biological sample to a capturematrix, repeated buffer exchanges, termed herein “washes,” are carriedout with the goal of removing cells weakly or non-specifically bound tothe capture matrix. Generally, cells are removed from the capture matrixand reapplied for every wash cycle. Removal and reapplication can beaccomplished manually by pipetting from tubes or automated using a pumpand tubing system. For example, using Quad Technologies MagCloudz®coupled with Dynabeads® magnetic cell separation system, cell-magneticparticle complexes are separated in tubes on a magnetic stand and washesare done manually. Using the Miltenyi Biotec CliniMACS® System, apre-set automated program applies the cell-magnetic particle complexesto a magnetic column in a tubing set and washes/reapplications are doneusing a valve pump system. In certain embodiments, selection may beperformed on various instruments, including without limitation theMiltenyi Biotec MACSQuant Tyto®, Quad Technologies MagCloudz®, GE Sepax®Cell Separation System, Terumo Elutra® Cell Separation System, COBESpectra® Cell Separator, SynGen LAB® or WASH® Systems, Fresenius-KabiLovo®, Miltenyi Biotec CliniMACS® System or CliniMACS Prodigy® System.Selection may be performed in a laboratory or at point-of-care. Detailedmethods for preparation and enrichment of cells and cell populations,including exemplary methods for selection of CD34+ cells are described,e.g., in Int'l Patent Pub. No. WO 2016/118790. Illustrative selectionmethod useful for high-stringency selection are provided by U.S. Pat.No. 8,727,132.

In certain embodiments, peripheral blood is obtained through a syringeor catheter inserted into a subject's or donor's vein. For example, theperipheral blood can be collected using an apheresis machine. Bloodflows from the vein through the catheter into an apheresis machine,which separates the white blood cells, including HSPC from the rest ofthe blood and then returns the remainder of the blood to the subject'sbody. Apheresis can be performed for several hours over successive days(e.g., 1 to 5 days) until enough HSPC have been collected.

In certain embodiments, bone marrow is obtained from the posterior iliaccrest of the subject by needle aspiration (see, e.g., Koda et al., 1984,J. Clin Invest. 73:1377-1384).

In certain embodiments, a hematocrit level of the biological sample maybe determined. The hematocrit level may be determined by centrifugingthe sample within a treatment chamber to separate RBCs of a sample intoa layer such that the packed cell volume may be determined. It should beappreciated that the sample may be combined with an anticoagulant inorder to assist with determining the hematocrit level and that such ananticoagulant may be added to the treatment chamber prior to or duringcentrifugation. Alternatively, the hematocrit level may be determined bymeasuring optical properties of the sample. For example, a spectrometermay be used to analyze the sample. It should be appreciated that anytype of known spectroscopic methods of determining hematocrit level maybe used such as, for example, Raman spectroscopy and/or light scatteringtechniques.

In certain embodiments, the biological sample is depleted oferythrocytes, e.g., before preparing the one or more cell populationsenriched for CD34+ cells from the biological sample. In someembodiments, the cells remaining after depletion techniques are washed.In another embodiment, non-specific IgG is added to the washed cells. Insome embodiments, the non-specific IgG is flebogamma.

Embodiments of the present invention comprise mammalian cells (e.g.,CD34+ cells) transduced with a viral delivery vector, e.g., a LV vectorcontaining the human PKLR gene. Accordingly, the present inventionincludes a method of transducing a mammalian cell, e.g. a humanhematopoietic stem cell or other cell described herein, comprisingcontacting the cell with a viral delivery vector, e.g., a LV vector,comprising an expression cassette described herein. In certainembodiments, the cell was previously obtained from a subject to betreated, or from another donor. In particular embodiments, the subjectwas diagnosed with PKD, and the cell is transduced with a LV comprisingan expression cassette encoding pyruvate kinase, e.g., a coRPK codingregion or cDNA. It is understood that the disclosed methods, e.g., thoseused to deliver a pyruvate kinase gene product, e.g., using a coPRK cDNAsequence, to a subject may also be used to treat hemolytic anemia,and/or normalize erythroid differentiation, increase the number offunctional mature erythrocytes, reduce extramedullar erythropoiesis,reduce splenomegaly and other secondary effects of hemolytic anemia orPKD.

To promote expression of the transgene, the subject polynucleotidecassette or gene delivery vector comprising a subject polynucleotidecassette will be contacted with the cells for about 30 minutes to 24hours or more, e.g., 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5hours 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 12 hours, 16 hours,18 hours, 20 hours, 24 hours, etc.

The subject polynucleotide cassette or gene delivery vector comprising asubject polynucleotide cassette may be provided to the subject cells oneor more times, e.g. one time, twice, three times, or more than threetimes, and the cells allowed to incubate with the agent(s) for someamount of time following each contacting event e.g. 16-24 hours, afterwhich time the media is replaced with fresh media and the cells arecultured further. Contacting the cells may occur in any culture mediaand under any culture conditions that promote the survival of the cells.The culture may contain growth factors to which the cells areresponsive. Growth factors, as defined herein, are molecules capable ofpromoting survival, growth and/or differentiation of cells, either inculture or in the intact tissue, through specific effects on atransmembrane receptor. Growth factors include polypeptides andnon-polypeptide factors.

Typically, an effective amount of subject polynucleotide cassette orgene delivery vector comprising a subject polynucleotide cassette isprovided to produce the expression of the transgene in cells. Asdiscussed elsewhere herein, the effective amount may be readilydetermined empirically, e.g. by detecting the presence or levels oftransgene gene product, by detecting an effect on the viability orfunction of the cells, etc. Typically, an effect amount of subjectpolynucleotide cassette or gene delivery vector comprising a subjectpolynucleotide cassette will promote greater expression of the transgenein cells than the same amount of a polynucleotide cassette as known inthe art. Typically, expression will be enhanced 2-fold or more relativeto the expression from a reference, or control, polynucleotide cassettee.g. as known in the art, for example 3-fold, 4-fold, or 5-fold or more,in some instances 10-fold, 20-fold or 50-fold or more, e.g. 100-fold.

For instances in which cells are to be contacted in vivo with a subjectpolynucleotide cassette or gene delivery vector comprising a subjectpolynucleotide cassette, the subject may be any mammal, e.g. rodent(e.g. mice, rats, gerbils), rabbit, feline, canine, goat, ovine, pig,equine, bovine, or primate. In preferred embodiments, the primate is ahuman. In some embodiment, the cells are CD34+ cells.

The methods and compositions of the present disclosure find use, e.g.,in the treatment of pyruvate kinase deficiency.

In another embodiment, the present invention includes a method oftreating a disease in a subject in need thereof comprising providing tothe subject an effective amount of cells transduced with a gene deliveryvector, e.g., a viral vector, that expresses a therapeutic gene productin the cells. In particular embodiments, the cells are autologous to thesubject. In certain embodiments, the cells are erythroid cells, e.g.,hematopoietic stem cells or committed hematopoietic erythroid progenitorcells. In some embodiments, the cell is a bone marrow cell, e.g., alineage depleted bone marrow cell. In particular embodiments, the methodis used to treat PKD, and the viral vector is a LV comprising anexpression construct disclosed herein comprising a human PGK promoteroperably linked to a coRPK gene cDNA or coding sequence, and a mutatedwPRE disclosed herein. In particular embodiments, the cells are providedto the subject parenterally, e.g., via intravenous injection. In someembodiments, the cells are provided to the subject by transfusion.

In another embodiment, the present invention includes a method oftreating PKD in a subject in need thereof, comprising providing to thesubject an effective amount of autologous CD34+ stem cells transducedwith a LV vector that expresses a coRPK cDNA in the cells, wherein theLV vector comprises a human PGK promoter operably linked to the coRPKcDNA or coding sequence, and a mutated wPRE sequence disclosed herein.In particular embodiments, the cells are hematopoietic stem cells orcommitted hematopoietic erythroid progenitor cells, e.g., bone marrowcells. In particular embodiments, the cells are provided to the subjectparenterally, e.g., via intravenous injection.

In another embodiment, the present invention provides a of treating adisease in a subject in need thereof comprising providing to the subjectan effective amount of a gene delivery vector, e.g., a viral vector,that expresses a therapeutic gene product in the subject. In particularembodiments, the method is used to treat PKD, and the viral vector is aLV comprising an expression construct disclosed herein comprising ahuman PGK promoter operably linked to a coRPK gene cDNA or codingsequence, and a mutated wPRE disclosed herein. In particularembodiments, the gene delivery vector are provided to the subjectparenterally, e.g., via intravenous injection.

In particular embodiments, the cells or gene delivery vectors areprovided to the subject in pharmaceutical compositions.

In some embodiments, the subject methods result in a therapeuticbenefit, e.g. preventing the development of a disorder, halting theprogression of a disorder, reversing the progression of a disorder, etc.In some embodiments, the subject method comprises the step of detectingthat a therapeutic benefit has been achieved. The ordinarily skilledartisan will appreciate that such measures of therapeutic efficacy willbe applicable to the particular disease being modified, and willrecognize the appropriate detection methods to use to measuretherapeutic efficacy.

Expression of the transgene using the subject transgene is expected tobe robust. Accordingly, in some instances, the expression of thetransgene, e.g. as detected by measuring levels of gene product, bymeasuring therapeutic efficacy, etc. may be observed two months or lessafter administration, e.g. 4, 3 or 2 weeks or less after administration,for example, 1 week after administration of the subject composition.Expression of the transgene is also expected to persist over time.Accordingly, in some instances, the expression of the transgene, e.g. asdetected by measuring levels of gene product, by measuring therapeuticefficacy, etc., may be observed 2 months or more after administration ofthe subject composition, e.g., 4, 6, 8, or 10 months or more, in someinstances 1 year or more, for example 2, 3, 4, or 5 years, in certaininstances, more than 5 years.

In certain embodiments, the method comprises the step of detectingexpression of the transgene in the cells or in the subject, whereinexpression is enhanced relative to expression from a polynucleotidecassette not comprising the one or more improved elements of the presentdisclosure. Typically, expression will be enhanced 2-fold or morerelative to the expression from a reference, i.e. a controlpolynucleotide cassette, e.g. as known in the art, for example 3-fold,4-fold, or 5-fold or more, in some instances 10-fold, 20-fold or 50-foldor more, e.g. 100-fold, as evidenced by, e.g. earlier detection, higherlevels of gene product, a stronger functional impact on the cells, etc.

Typically, if the subject composition is an LV comprising the subject apolynucleotide cassette of the present disclosure, an effective amountto achieve a change in will be about 1×10⁸ vector genomes or more, insome cases 1×10⁹, 1×10¹⁰, 1×10¹¹, 1×10¹², or 1×10¹³ vector genomes ormore, in certain instances, 1×10¹⁴ vector genomes or more, and usuallyno more than 1×10¹⁵ vector genomes. In some cases, the amount of vectorgenomes that is delivered is at most about 1×10¹⁵ vector genomes, e.g.1×10¹⁴ vector genomes or less, for example 1×10¹³, 1×10¹², 1×10¹¹,1×10¹⁰, or 1×10⁹ vector genomes or less, in certain instances 1×10⁸vector genomes, and typically no less than 1×10⁸ vector genomes. In somecases, the amount of vector genomes that is delivered is 1×10¹⁰ to1×10¹¹ vector genomes. In some cases, the amount of vector genomes thatis delivered is 1×10¹⁰ to 3×10¹² vector genomes. In some cases, theamount of vector genomes that is delivered is 1×10⁹ to 3×10¹³ vectorgenomes. In some cases, the amount of vector genomes that is deliveredis 1×10⁸ to 3×10¹⁴ vector genomes.

In some cases, the amount of pharmaceutical composition to beadministered may be measured using multiplicity of infection (MOI). Insome cases, MOI may refer to the ratio, or multiple of vector or viralgenomes to the cells to which the nucleic may be delivered. In somecases, the MOI may be 1×10⁶. In some cases, the MOI may be 1×10⁵-1×10⁷.In some cases, the MOI may be 1×10⁴-1×10⁸. In some cases, recombinantviruses of the disclosure are at least about 1×10¹, 1×10², 1×10³, 1×10⁴,1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸, 1×10⁹, 1×10¹⁰, 1×10¹¹, 1×10¹², 1×10¹³,1×10¹⁴, 1×10¹⁵, 1×10¹⁶, 1×10¹⁷, and 1×10¹⁸ MOI. In some cases,recombinant viruses of this disclosure are 1×10⁸ to 3×10¹⁴ MOI. In somecases, recombinant viruses of the disclosure are at most about 1×10¹,1×10², 1×10³, 1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸, 1×10⁹, 1×10¹⁰, 1×10¹¹,1×10¹², 1×10¹³, 1×10¹⁴, 1×10¹⁵, 1×10¹⁶, 1×10¹⁷, and 1×10¹⁸ MOI.

In some aspects, the amount of pharmaceutical composition comprisesabout 1×10⁸ to about 1×10¹⁵ particles of recombinant viruses, about1×10⁹ to about 1×10¹⁴ particles of recombinant viruses, about 1×10¹⁰ toabout 1×10¹³ particles of recombinant viruses, or about 1×10¹¹ to about3×10¹² particles of recombinant viruses.

Any total number of viral particles suitable to provide appropriatetransduction of cells to confer the desired effect or treat the diseasecan be administered to the mammal. In various preferred embodiments, atleast 10⁸; 5×10⁸; 10⁹; 5×10⁹, 10¹⁰, 5×10¹⁰; 10¹¹; 5×10¹¹; 10¹²; 5×10¹²;10¹³; 1.5×10¹³; 3×10¹³; 5×10¹³; 7.5×10¹³; 9×10¹³, 1×10¹⁴ viralparticles, or 5×10¹⁴ viral particles or more, but typically not morethan 1×10¹⁵ viral particles are injected. Any suitable number ofadministrations of the vector to the mammal or the primate eye can bemade. In one embodiment, the methods comprise a single administration;in other embodiments, multiple administrations are made over time asdeemed appropriate by an attending clinician. In some embodiments atleast 2×10⁸ VG/ml of 5×10⁵ cells/ml is required in a singleadministration (24 hours transduction) to result in high transductionefficiencies. Individual doses are typically not less than an amountrequired to produce a measurable effect on the subject, and may bedetermined based on the pharmacokinetics and pharmacology forabsorption, distribution, metabolism, and excretion (“ADME”) of thesubject composition or its by-products, and thus based on thedisposition of the composition within the subject. This includesconsideration of the route of administration as well as dosage amount.Effective amounts of dose and/or dose regimen can readily be determinedempirically from preclinical assays, from safety and escalation and doserange trials, individual clinician-patient relationships, as well as invitro and in vivo assays such as those described herein and illustratedin the Examples.

Several aspects of the invention are described herein with reference toexample applications for illustration. It should be understood thatnumerous specific details, relationships, and methods are set forth toprovide a full understanding of the invention. One having ordinary skillin the relevant art, however, will readily recognize that the inventioncan be practiced without one or more of the specific details or withother methods. The present invention is not limited by the illustratedordering of acts or events, as some acts may occur in different ordersand/or concurrently with other acts or events. Furthermore, not allillustrated acts or events are required to implement a methodology inaccordance with the present invention.

It is further noted that the claims may be drafted to exclude anyoptional element. As such, this statement is intended to serve asantecedent basis for use of such exclusive terminology as “solely”,“only” and the like in connection with the recitation of claim elements,or the use of a “negative” limitation.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application.Nothing-herein is to be construed as an admission that the presentinvention is not entitled to antedate such publication by virtue ofprior invention. Further, the dates of publication provided may bedifferent from the actual publication dates which may need to beindependently confirmed.

All of the above U.S. patents, U.S. patent application publications,U.S. patent applications, foreign patents, foreign patent applicationsand non-patent publications referred to in this specification and/orlisted in the Application Data Sheet, are incorporated herein byreference, in their entirety, e.g., to disclose and describe the methodsand/or materials in connection with which the publications are cited. Itis understood that the present disclosure supersedes any disclosure ofan incorporated publication to the extent there is a contradiction.

From the foregoing it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

Experimental Methods

Vectors and LV supernatant production. LVs were generated as describedherein. CoRPK sequence was designed using the GeneArt® software toincrease the GC content of the sequence and to prevent cryptic splicesites. Vectors were developed using the pCCL.sin.ppt.hPGK-EGFP-wPRE*construct as a backbone, generously provided by Dr. Naldini (HSR-TIGET,San Raffaele Telethon Institute, Milano, Italy). The original pCCLlentivirus transfer vector and its elements are described in Dull etal., J Virol, 1998; 72(11): 8463-8471, which is incorporated herein inits entirety. Vector stocks of VSV-G pseudotyped LVs were prepared by3-plasmid calcium phosphate-mediated transfection in 293T cells (ATCC:CRL-1573, Rockeville, MD, USA), as previously described [Follenzi A, etal. (2000). Nat Genet 25: 217-222]. Titers of infective LVs weredetermined in HT1080 cells (ATCC: CCL-121) by qPCR as describedelsewhere [Charrier S, et al. (2005). Gene Ther 12: 597-606]. LV stocksof 10⁷-10⁸ viral particles (vp)/mL titers were routinely obtained.

Purification and transduction of murine HSCs. BM from 8-14 week-old malePKD mice was harvested from the leg bones and lineage negative cells(Lin⁻) were purified using the Lin⁻ Cell Depletion kit (Miltenyi Biotec,Gladbach, Germany), obtaining 70-90% purity. Lin⁻ cells werepre-stimulated with 100 ng/mL of recombinant human IL-11 (Peprotech ECLtd., London, UK) and 100 ng/mL of recombinant murine SCF (R&D SystemsInc., Minneapolis, MN) in IMDM-Glutamax medium supplemented with 20% FBSand 0.5% antibiotics (50 U/mL penicillin and 50 μg/mL streptomycin,(Thermo Fisher Scientific, Waltham, MA) for 24 h, and then transducedwith EGFP or coRPK carrying LVs in two cycles of transduction at MOIs of1-10 vp/cell. Each transduction was carried out for 24 h in the presenceof the aforementioned cytokines on plates previously coated with CH-296fibronectin fragment (2 μg/cm²; Retronectin, TakaraShuzo, Otsu, Japan)overnight at 4° C.

In vivo RBC survival. Transplanted mice carrying the coRPK transgenewere injected with three consecutive intravenous injections (12 h apart)of Biotin 3-sulfo-N-hydroxysuccinimide ester sodium salt (50 mg/kg)(Sigma Aldrich, Saint Louis, MO). Twelve hours after the last injection,tail vein blood was harvested and labelled with 2 μg/mL of anti-mouseTer119-PE (BD Bioscience, San Jose, CA) and streptavidin-FITC (50 μg/mL,BD Biosciences, San Jose, CA) for 30 min at 4° C. Samples were analyzedin an EPICS XL flow cytometer (Beckman Coulter, Brea, CA) every 2-4 daysfor 40 days after the injection. RBC survival kinetics was measured bythe percentage of biotinylated cells within the total RBC population.

CFC Assay. CFC assay was performed in BM and spleen from control andtransplanted mice according to manufacturer's procedure from Methocultmedium GF M3434 (Stem Cell Technologies, Vancouver, Canada). BM cellswere harvested at different time-points after transplant from all groupsof mice, and CFUs (clusters of 30 or more cells) were scored 7 daysafter seeding in a Nikon Diaphot-TMD microscope.

Identification of hematopoietic lineages. PBMCs were obtained from thetail vein of transplanted animals and labelled with a panel ofantibodies to detect different hematopoietic cells. Myeloid cells weredetected with anti-GR-1 and anti-Mac-1 biotinylated antibodies (BDBioscience, San Jose, CA, 5 μg/mL), while lymphoid cells were detectedusing anti-CD3-PE antibody for T-cells, and anti-B220-PE andanti-B220-PECy5 antibodies for B-cells (BD Bioscience, San Jose, CA, 10μg/mL), together with SAV-TRC secondary antibody (Invitrogen, ThermoFisher Scientific, Waltham, MA). Samples were analysed in a BD LSRFortessa Cytometer (BD Bioscience, San Jose, CA, USA) adding DAPI(Boehringer, Ingelheim, Germany, 2 μg/mL) to exclude death cells.

Structural and histological studies. Spleens were collected,photographed and weighed on precision scales to determine the presenceof splenomegaly. Histological studies were performed on spleen and liversections obtained following conventional histological methods, andstained with hematoxylin (Gill-2 Haematoxylin, Thermo, Pittsburgh, USA)and eosin (Eosin Alcoholic, Thermo Fisher Scientific, Waltham, MA). Irondeposits were also studied in the spleen by Prussian Blue or Perls'staining (Sigma Aldrich, Saint Louis, MO) following manufacturer'sinstructions. All sections were examined using an Olympus BX40 lightmicroscope and photographed with an Olympus DP21 camera, with a finalmagnification of 100× or 200×.

Erythroid differentiation. Flow cytometry analysis of Ter119 and CD71marker intensities in BM and spleen were used to identify the differenterythroid subpopulations as described elsewhere [Socolovsky M, et al.(2001). Blood 98: 3261-3273] using 4 μg/mL of anti-mouse Ter119-PEantibody (BD Bioscience, San Jose, CA,), 10 μg/mL of biotinylatedanti-CD71 antibody (BD Bioscience, San Jose, CA,) andstreptavidin-tricolor (Invitrogen, Thermo Fisher Scientific, Waltham,MA). Cells were then analyzed in an EPICS XL flow cytometer (BeckmanCoulter, Brea, CA) using propidium iodide (IP, 2 μg/mL) to detect livecells.

Provirus quantification. Detection and quantification of integratedprovirus per cell was accomplished using complementary primers to thepackaging proviral sequence (Ψ) and the mouse Titin housekeeping gene.Total BM and peripheral blood samples were collected periodically, andgenomic DNA from nucleated cells was isolated using the DNeasy Blood &Tissue kit (Qiagen, Venlo, Limburg, The Netherlands). Twenty to 50 ng ofgenomic DNA (gDNA) were amplified by multiplex qPCR using the 7500 FastReal-Time PCR System (Applied Biosystems, Thermo Fisher Scientific,Waltham, MA) and primers and probes previously described [Charrier S, etal. (2011). Gene Ther 18: 479-487].

Chimerism. Presence of donor cells was quantified by qPCR detecting theY chromosome SRY gene and the mouse β-Actin housekeeping gene. Primersand probes previously described [Navarro S et al (2006). Mol Ther 14:525-535] were used and genomic DNA from PB of transplanted mice wasamplified using the 7500 Fast Real-Time PCR System (Applied Biosystems,Thermo Fisher Scientific, Waltham, MA). Standard curves were preparedusing gDNA extracts from samples containing 0% to 100% of BM cells frommale/female mouse mixtures and chimerism was calculated as: % of donorengraftment=100×2^((CtβAct-CtSRY)).

LAM-PCR procedure. In order to identify vector integration sites, 3′vector LTR-genome junctions were amplified by LAM-PCR following themethod published by Schmidt et al. 2007 [Nat Methods 4: 1051-1057]. Thestarting linear amplification (100 cycles) was performed usingbiotinylated LTR specific primers and up to 100 ng of gDNA as template.Linear amplification products were purified using streptavidin magneticbeads and followed by complementary strand synthesis, parallel digestionwith 2 different restriction enzymes (Tsp509I and HpyCH4IV) and twoligation reactions using linker cassettes complementary to the ends leftby the enzyme's cut. The fragments generated were amplified by twoadditional exponential PCR steps. LAM-PCR products were separated andquantified by gel electrophoresis on a MultiNA automated system(Shimadzu).

Setup of LAM-PCR products for Illumina MiSeq sequencing. Following themethod published by Parazynski et al [Paruzynski A, et al. (2010). NatProtoc 5: 1379-1395], 40 ng of the second exponential PCR productsgenerated by Tsp509I and HpyCH4IV enzymes were re-amplified using fusionprimers containing specific sequences that allow paired end sequencingon an Illumina MiSeq sequencer. LAM-PCR samples were adapted for454-pyrusequencing by fusion PCR to add the Roche 454 GS-FLX adaptors:adaptor A plus an 8-nucleotide barcode was added to the LTR end of theLAM-PCR amplicon; adaptor B was added to the linker cassette side. In5′-3′ orientation the final amplicon was composed as follow: adaptor A,barcode, LTR sequence, unknown genome sequence, linker cassette sequenceand primer B. Purified fusion primer PCR products were run andquantified on a MultiNA automated electrophoresis system, and pooledtogether in order to obtain a final equimolar library of 10 nM. Thefinal library was then re-quantified using a KAPA Library QuantificationKit for Illumina Sequencing Platform (Kapa Biosystems, Wilmington, MA)on a Viia7 real-time PCR system (Applied Biosystems, Thermo FisherScientific, Waltham, MA), obtaining an estimated concentration of 16.35nM. Finally, libraries were sequenced using the Illumina MiSeq ReagentKit.

Bioinformatics analysis. To extract vector IS from a high-throughputsequencing platform, both Roche 454 and Illumina MiSeq/HiSeq, a pipelinetaking in input the row data (typically in FastQ file format) wasdesigned, providing the list of reliable IS and the nearest gene.Superior level analyses for clonal abundance quantification and geneontology enrichment were performed using Excel, GraphPad Prism (TM) andavailable online tools.

NGS data processing and pipeline usage. The step of NGS data processingdeals with the management of high-throughput data from Illumina MiSeqsequencing platforms and aims at identify IS in which all valid sequencereads are aligned to the reference genome. Data processing comprises twomain activities: 1. Data quality inspection and analysis, in which LVvector sequences and other contaminants are trimmed. 2. Integration siteidentification, in which all valid sequence reads are aligned to thegenome of reference and valid ISs are retrieved.

Data quality analysis. In order to identify IS from Illumina MiSeq rawdata a bioinformatics pipeline was developed. Standard LAM-PCR productscontain a LTR sequence, a flanking human genomic sequence and a linkercassette (LC) sequence. The 459 technology allowed retrieval of LAM-PCRsequences with length ranging from 10 bp to 900 bp. Similar results wereretrieved from Illumina MiSeq paired-ends reads. These length boundariesare important parameters to consider in the quality analysis processsince they affect both, the subsequent alignment procedure and thealgorithm of the vector components identification. Sequences too shortto be correctly aligned to the reference gene were discarded, as well asthose exceeding the maximum size reachable with NSG technology to avoidmissing part or all of the LC sequence. Once the pipeline ends for eachpool, all integration sites were collected both in files (archived inthe TIGET network attached file storage -NAS-) and in the internaldatabase, and maintained in a storage server that keeps track of themodified copies.

Integration site identification. To identify unique integration sitesand extract the excel file with all IS in rows and each sample incolumns (IS matrix) with the closest gene annotations, we run thefollowing steps: 1. Creation of the IS matrix using the program calledcreate matrix, enabling the collision detection inter projects. Thisprogram will produce a tab-separated file (TSV); 2. Annotation of the ISmatrix file using the program annotate_bed, that will be called asfollows for each pool using the input TSV file: awk ‘{print “chr” $1“\t” $2 “\t” $2}’ TSV_FILE|tail −n+2>TSV_FILE.bed;annotate_bed—a/opt/genome/mouse/mm9/annotation/mm9.refGene.TIGET.gtf—bTSV_FILE.bed—o TSV_FILE.annotatedbed; 3.Import both annotation andmatrix file into a new Excel worksheet, here on called XLS.

Collision detection. In order to obtain a reliable dataset of ISs fromeach transplanted mouse, we filtered data from potentialcontaminations/collisions and from false positives based on sequencecounts. An additional step of data normalization was required to combineintegration sites resulting from different experiments.

The term “collision” is used to identify the presence of identical IS inindependent samples. In our experimental setting, the integration ofvector in the very same genomic position in different cells is a verylow probability event. Thus, the detection of identical ISs inindependent samples likely derives from contamination, which may occurat different stages of wet laboratory procedures (sample purification,DNA extraction, LAM-PCRs and sequencing). Although our working pipelineis designed to minimize the occurrence of inter-samples contacts, thehigh-throughput analysis of ISs intrinsically carries a certain degreeof background contamination. Identification of the extent ofcontamination between samples is crucial also because the retrieval ofthe same IS in different samples obtained from the same mouse is used insubsequent steps to make inference on biological properties of thevector-marked hematopoietic cells (i.e. multi-lineage potential andsustained clonogenic activity). Thus, we must be able to distinguish theactual occurrence of the same IS in different samples (from the samemouse) from a contamination/collision.

To address these issues, we assessed the extent of shared IS amongsamples derived from different test items and mice as a way to measurethe extent of collision in our analyses and then design rules to discardfrom each mouse's data set those IS that can be ascribed to collisionand minimize the likelihood of scoring false positive when looking forshared IS between samples from the same mouse. We designed a collisiondetection process allowing the validation of each integration locus. Theoverall result should be that, given the set I of integration loci, incase of classification of an integration locus i in I as collision, i isdiscarded from I We applied collision detection process between 3independent transplantation groups: 1. coPKR170s: mice from assay 1euthanized at 170 days after transplant with Lin⁻ cells transduced withthe coRPK expressing LV vector (coRPK 1-3). 2. EGFP: mice from assay 2transplanted with Lin⁻ cells carrying the EGFP expressing LV vector(EGFP 1-6). 3.coPKR-TC: mice from assay 2 transplanted with Lin⁻ cellstransduced with the coPKR expressing LV vector (coRPK 1-14), whose bloodand BM was analysed at different time-points, including secondaryrecipients transplanted with a pooled BM from a sub-group of primarytransplanted mice (coRPK 11-14).

Each identical IS has different sequence reads (sequence count) amongthe different mice. Sequence counts can be used to determine whethersamples from one mouse contaminated the other mice' samples based on theabundance criterion. In our rationale, an integration found in two micewill be assigned to the mouse that shows the highest abundance, while inthe other mouse it will be considered as a contaminant. Therefore, wecould identify a threshold of differential sequence count that allowsassigning a given collision to a mouse and removing from the others. Weretrieved the threshold value from our data obtaining a value of 10,meaning that for each IS, among all TI, if an IS has got an abundancevalue (percentage sequence count ratio) 10 times lower than the highestabundance value (percentage sequence count) of the other TIs, then it isdiscarded from the current TI. We applied these rules both among the TIand the selected groups, and Excel files were used to compute thecollisions detection by applying the following rules (here detailed forTI filtering but that are extended to groups filtering as well): 1.Isolating each TI, group all samples of the same TI together by summingthe sequence counts. 2. For the three TIs obtained, for each IS, computethe percentage ratio of the IS sequence count versus the overall sum ofreads for the TI. 3. Then, applying the following rule to compute thedecision step with the threshold of 10 that allowed to assigning each ISto a reliable TI.

Once an IS was detected to remove, reads of that IS were removed fromthe group so that it will no longer assigned also to that group. Thefilter described above was applied between the mice transplanted withdifferent ex-vivo transduced cell populations (one cohort of EGFPexpressing mice from assay 2, and two cohorts of coPKR mice belonging totwo independent transplantation experiments). Moreover for the coPKR-TCgroup (assay 2), the filter method mentioned above was modified in twoways: a) To better highlight the sharing of integrations betweentime-points in the context of the clonal abundance analysis we added thefollowing rule: if one integration is shared between one or more micethen the integration will be kept for all time points even if theirsequence count is less than the 10% of the maximum sequence count amongmice; b) For lineage tracking relationships we applied a more stringentfilter by eliminating the IS with a sequence count lower than 3 and the10% sequence count filter for sharing between time-points. Meaning thatan integration shared between two time points will be kept or discardedonly if is more or less than the other respectively.

Gene ontology analysis. All gene ontology analyses were made using theGREAT online software (http://bejerano.stanford.edu/great/public/html/).The web page allow to upload the genomic coordinates of the integrationsof each dataset and calculates the enrichment levels in the testeddataset by correlating positional information (based on the binomialdistribution analysis for p-value calculations) and annotated functionof the genes nearest to the integration sites (based on thehypergeometric distribution analysis for p-value calculations)[Groeschel S, et al. (2011). J Inherit Metab Dis 34: 1095-1102].Biological processes and molecular functions of the GO database werechosen for enrichment analysis. Only the gene classes with a falsediscovery rate <0.05 for both statistical analyses were considered (FIG.20 ).

Data storage. All data, both row data and results, are stored in TIGETnetwork attached file storage (NAS) in the root folder, in which allalignments from the pipeline are available, as well as the abundancematrix and plots. NAS storage is secured by authentication andauthorization policies, and was built on a reliable and scalableinfrastructure using redundancy array of disks RAID 5, and it is underbackup on our CrashPlan software registered in TIGET.

Example 1 PGK-coRPK Therapeutic LV Vector Leads to a Stable andLong-Term Correction of the Anemic Phenotype in Genetically CorrectedPKD Mice

In vivo efficacy of the PGK-coRPK LV (FIG. 2 a ) was assayed bytransduction and transplantation of lineage depleted BM cells (Lin⁻cells) from PKD mice (FIG. 2 b ). FIG. 2 a is a schematic representationof the SIN LV vectors used throughout gene therapy experiments harboringthe human PGK promoter regulating the expression of the EGFP transgenein the control vector (upper diagram) or the expression of coRPK cDNA inthe therapeutic vector (lower diagram). The coRPK sequence showed 80.4%homology with the human PKLR cDNA and 76.5% homology with the mouse PklrcDNA, with no changes in the amino acid sequence. FIG. 2 b is aschematic of the gene therapy protocol performed to address thefunctionality of the developed PGK-coRPK LV vector. Correction of thePKD phenotype was studied for 4 to 9 months after transplant in PB andBM through hematological analysis and metabolic profiling. Integrationanalysis was performed in different tissues and time-points from allmice to address LV vector safety. At 280 days post-transplantation,total BM from primary transplanted mice carrying the coRPK transgene wastransplanted again into lethally-irradiated female PKD mice (secondaryrecipients) to test the stability and safety of the engraftment.Lethally irradiated PKD mice transplanted with deficient cellstransduced with the coRPK LV showed a significant improvement in alltested blood erythroid parameters when compared to non-transplanted PKDlittermates or to mice transplanted with cells transduced with an EGFPLV (FIG. 3 and Table 2).

TABLE 2 Hematological variables recorded 140 days post-transplantationin peripheral blood. Group HGB (g/dL) HTC (%) MCV MCH (pg) Healthy (n =5) 14.64 ± 0.99  36.15 ± 2.64 38.20 ± 0.86 15.44 ± 0.21 PKD (n = 9) 9.70± 0.55 28.91 ± 1.53 51.56 ± 0.50 17.23 ± 0.31 EGFP (n = 8) 6.81 ± 0.6821.32 ± 1.51 49.13 ± 0.72 15.46 ± 0.62 coRPK (n = 17) 10.67 ± 0.53* 31.09 ± 1.45* 45.65 ± 0.84 15.35 ± 0.53 2nd coRPK (n = 4)  12.40 ±0.67**  34.11 ± 1.49* 46.25 ± 0.85 16.78 ± 0.23

Data represent the mean±SEM and were statistically analysed bycomparison to EGFP-expressing mice using the Kruskal-Wallisnon-parametric test. *p<0.05; **p<0.01. RBC counts increased as soon as40 days post-transplantation (FIG. 3 a ) and constitutivereticulocytosis, which is one of the most common signs of PKD, wassignificantly reverted in mice carrying the PGK-coRPK transgene,reaching levels close to those observed in healthy controls for at least9 months after transplant (FIG. 3 b ). On the contrary, PKD animalstransplanted with EGFP LV-transduced cells showed anemia and remarkablereticulocytosis in parallel with PKD mice at all time-points analyzed.Hemoglobin levels (HGB), hematocrit index (HTC), mean corpuscular volume(MCV) and mean corpuscular hemoglobin (MCH) values were also correctedin mice transplanted with LV-corrected cells when compared tonon-transplanted PKD littermates (Table 2). This hematologicalcorrection was achieved with 63.66±4.45% of donor chimerism andtransduction efficacies ranging from 60% to 90% (Table 3).

TABLE 3 Relevant molecular parameters in mice transplanted withgenetically modified cells. Donor Transduction chimerism % % Vector copynumber (VCN/cell) Provirus⁺ SRY⁺ PB Assay Groups WBC Total BM CFU CFUscells 1 EGFP (n = 2) 0.83 ± 0.05 0.42 ± 0.03 0.42 ± 0.00 57.73 ± 12.28n.d MOI coRPK (n = 3) 4.76 ± 0.28 3.58 ± 0.34 3.07 ± 0.76 91.08 ± 3.67 n.d 1-4 2 EGFP (n = 6) 4.56 ± 0.50 4.19 ± 1.29 3.93 ± 0.98 92.32 ± 7.68 61.82 ± 3.61 MOI coRPK (n = 14) 1.65 ± 0.08 0.99 ± 0.13 1.89 ± 0.4262.06 ± 11.73 63.66 ± 4.45 10 2^(nd) coRPK 1.44 ± 0.08 n.d n.d 63.15 ±0.31^(a ) 62.89 ± 5.61 (n = 4) Data represent the mean ± SEM, n.d, notdetermined. ^(a)estimated transduction percentage obtained byinterpolation in the linear regression built from experiment 1 (X axis:VCN/WBC, Y axis: % provirus⁺ CFUs).

Transduced cells showed on average 1.65±0.08 integrated vector copiesper cell, indicating that PGK-coRPK LV-vector provided enough human RPKtransgenic expression to revert the hemolytic anemia. Remarkably, theexpression of the coRPK transgene led to an extension of erythroid cellhalf-life when compared to non-transplanted PKD mice (FIGS. 3 c,d ). Onaverage, PKD mice showed a RBC half-life of 19 days, while ingenetically corrected mice this was extended to 25 days (6 days'extension) reaching values close to wild-type RBC half-life (FIG. 3 d ).Thus, RBCs of coRPK-expressing mice displayed intermediate survivalkinetics between healthy and deficient control mice (FIG. 3 c ), mostlikely because full chimerism was not achieved in these animals (Table3).

Nine months after transplant hematopoietic progenitors from primaryrecipients were transplanted into secondary recipients that maintainedengraftment levels (62.89±5.61%) and VCN (1.44±0.08 copies) (Table 3).Secondary transplanted recipients showed a multi-lineage hematopoieticreconstitution up to 5 months post-transplantation (FIG. 4 ) and asignificant improvement in all PB erythroid parameters (FIG. 5 and Table2). FIG. 4 a is a diagram of the flow cytometry strategy used toidentify the different hematopoietic lineages by labeling with CD3-PE,B220-PE, B220-PECy5, Grl-Biotin and Macl-Biotin antibodies plusSAV-PE-Cy5. FIG. 4 b depicts representative dot-plots and percentages(FIG. 4 c ) of each lineage in PB at 140 days after transplant. Barsrepresent the average percentage±SEM of healthy (n=2, black bar) and PKDmouse (n=2, grey bar) controls and secondary transplanted miceexpressing the coRPK therapeutic transgene (n=4, scratched bar). Inaddition, proviral integrations were detected in differently committedhematopoietic progenitors (FIGS. 6 a,b ) and its number remainedconstant over time (FIG. 6 c ) demonstrating the stability of thegenetic correction and highlighting the safety of the PGK-coRPK LV. FIG.6 a shows the vector copy number per cell in BM CFUs from individualtransplanted mice at 120 to 170 days after transplant. Transduction andchimerism percentages are also shown. FIG. 6 b shows provirus copynumber in cells from different hematopoietic compartments. Columnsrepresent the average±SEM of the different groups of transplanted mice.FIG. 6 c shows the kinetics of proviral integrations in BM cells fromindividual transplanted EGFP-expressing mice (grey lines) and micecarrying the coRPK transgene (black lines).

Example 2 LV-Derived RPK Expression Normalizes Erythroid Differentiationand Allows the Production of Functional Mature Erythrocytes

PKD mice show a characteristic expansion of the erythroid compartmentcaused by the compensatory erythropoiesis mechanism (Min-oo et al 2004).The study of the erythroid differentiation pattern in transplanted miceindicated that the ectopic RPK expression reverted this mechanism (FIGS.7 a,b ). PKD and EGFP-expressing mice showed a predominance of immatureerythroid precursors (subpopulation I: proerythroblasts andsubpopulation II: basophilic erythroblasts) in BM and spleen, and aremarkable fall in late erythroid cells (population IV: reticulocytesand mature erythrocytes), while mice transplanted with cells transducedwith the coRPK LV showed a significant reduction of immature erythroidprecursors (subpopulations I and II) in BM and spleen, and a significantincrease of the latest erythroid compartment (subpopulation IV)equivalent to healthy mice (FIGS. 7 a,b ). In addition, unlike PKD andEGFP-expressing mice, those carrying the coRPK transgene showed asignificant reduction of erythropoietin (Epo) levels in plasma (FIG. 7 c). FIG. 8 a shows the total CFUs from spleen and FIG. 8 b shows the bonemarrow at 140 days after transplant. Dots represent number of coloniesper mouse analyzed and lines represent average±SEM in each group. Datawere statistically analyzed by non-parametric Kruskal-Wallis test. Thenormalization of the erythropoiesis in PKD mice treated with thetherapeutic vector was accompanied by a reduction of the splenic numberof progenitors content to normal levels (FIG. 8 a ), although no changesin the BM CFU content were noted (FIG. 8 b ).

Example 3 Transplantation of Cells Transduced with the coRPK LV VectorReverts Extramedullar Erythropoiesis and Organ Pathology

Due to the active destruction of RPK deficient erythrocytes, PKD andEGFP-expressing mice displayed acute splenomegaly with an increase ofspleen weight and size of over 200% when compared to healthy controls(FIGS. 9 a,b ). A disorganized structure of the splenic tissue and theexpansion of the spleen red pulp was also observed in these animals,indicating an intense extramedullar erythropoiesis also supported by thepresence of erythroid cell clusters in PKD and EGFP-expressing liversections (FIG. 9 c ). Remarkably, the ectopic expression of coRPKtransgene completely reverted spleen and liver pathology in geneticallycorrected mice, reducing the RBC accumulations and normalizing spleenhistological structure and size (FIG. 9 ). Additionally, histologicalstudies revealed the total absence of iron deposits in the liver ofgenetically corrected mice, whereas PKD mice either from thenon-transplanted group or the group transplanted with HSCs transducedwith the EGFP-carrying vector displayed an intense iron overload due tothe continuous hemolytic process (FIG. 9 c ). Overall, the transplant ofgenetically corrected HSCs in PKD mice restored the normal status of theerythropoiesis and all the secondary effects caused by the hemolyticanemia.

Example 4 PGK-coRPK LV-Derived Expression Restores the GlycolysisPathway in RBCs without Modifying the WBC Metabolic Balance

Next, we performed an extensive metabolomic analysis of all transplantedand control mice to study the functional correction of RPK enzymaticactivity. Following an untargeted profiling strategy, we observedsignificant changes of glycolytic intermediates in RBCs among thedifferent groups, identifying three broad clusters of metabolitepatterns with distinct trends (FIG. 10 a ). RBCs from coRPK-expressingmice showed an increase of metabolites from cluster 1 similar to healthycontrols but different from transplanted mice carrying the EGFPtransgene. Likewise, cluster 3 reflected a reduced metabolite trend ingenetically corrected mice similar to wild type mice and different fromEGFP-expressing mice. Nevertheless, cluster 2 from assay 1 showed nodifferences of metabolite profile between transplanted mouse groups(EGFP and coRPK expressing mice) (FIG. 10 a ). The untargeted metabolicprofiling also showed that the genetic modification was capable ofmodifying some important glycolytic intermediates, achieving an increasein ATP (FIG. 10 b ), ADP (FIG. 10 c ) and pyruvate (FIG. 10 d ) levelsin erythrocytes isolated from mice transplanted with PGK-coRPKLV-transduced HSCs. Considering these metabolic trends, we then analysedother metabolites located closer to the PK-catalysed reaction using atargeted profiling approach. Levels of the direct PK substratephosphoenolpyruvate (PEP) (FIG. 10 e ) and the 3-phosphoglycerate (3-PG)(FIG. 10 f ), located upstream of the PK-catalysed reaction, approachedto that of healthy control mice. Deficient erythrocytes expressing thecoRPK transgene also produced an increase of D-lactate (FIG. 10 g ), thefinal product of the anaerobic glycolysis, when compared to PKD and EGFPexpressing mice. To test whether the compensation in the glycolyticmetabolites was a result of the normalization in the PK activity inmature erythrocytes, we measured the activity of this enzyme andnormalized it in relation to the Hexokinase activity in order to avoidthe influence of high amounts of reticulocytes in the deficient animals.RBCs were purified through a cellulose column to prevent leukocyte PKactivity contamination. A complete compensation of PK activity wasobserved in the animals expressing the coRPK that reach ratios similarto those obtained from wild type healthy animals and from a normalhealthy blood donor volunteer (FIG. 11 ). FIG. 11 a shows pyruvatekinase activity, FIG. 11 b shows hexokinase activity and FIG. 11 c showsratio of pyruvate kinase and hexokinase enzymatic activities in RBCsfrom control mice and mice transplanted with transduced cells. RBCs werepurified from blood samples through a cellulose column to avoidleukocyte PK activity contamination and subjected to enzyme activityevaluation. Black bars, healthy mice (n=2); white bars, micetransplanted with cells transduced with the EGFP expressing vector(n=3); scratched bars, mice transplanted with cells transduced with thecoRPK expressing vector (n=3). Checkered bars represent values from ahealthy volunteer (n=1). Data represent the average±SEM of each group.

Principal component analysis showed that metabolite pattern of RBCs wasdifferent depending on the group and markedly different to WBC profile(FIG. 12 a ). On the contrary, WBC sub-groups clustered together withvery little difference among groups, indicating no changes in themetabolic balance of leukocytes when expressing the ectopic coRPK (FIG.12 a ). Additionally, specific metabolite changes observed in the RBCuntargeted profiling were not present in WBCs (FIGS. 12 b-d ).

Example 5 PGK-coRPK LV Transduced Cells Render Polyclonal HematopoieticReconstitution without Evidence of Vector Genotoxicity

Integration profile of LVs carrying either the coRPK or the EGFPtransgene was analysed in transplanted mice. Resulting from thegenome-wide integration profile of LVs, each insertion created a uniquegenetic mark that can be used to track the clonal behaviour inindividual transduced cells. Genomic DNA (gDNA) was obtained from WBCsand from BM cells and from primary and secondary transplanted mice, aswell as from transduced cell pools before transplant (Lin⁻ cells).Linear Amplification Mediated PCR (LAM-PCR) (FIGS. 13 and 14 ) was usedto amplify vector/genome junctions, and to identify vector ISs. FIG. 13demonstrates vector ISs were identified by LAM-PCR amplification of 3′vector LTR-genome junctions. A MultiNA automated system was used,generating a pattern characterized by several bands. Vector backbonederived Tsp509I internal control band (IC) is indicated by an arrow.FIG. 14 demonstrates vector ISs were identified by LAM-PCR amplificationof 3′ vector LTR-genome junctions. A MultiNA automated system was used,generating a pattern characterized by several bands. Vector backbonederived HpyCH4IV5 IC is indicated by an arrow.

PCR products were sequenced by MiSeq Illumina platform and the obtainedsequences were mapped onto the mouse genome by bioinformatics pipelineand filtered for collisions as described in the Methods section above(FIG. 15 ). FIG. 15 is the general scheme of the analysis of IS mappingperformed in mice transplanted with genetically modified hematopoieticprogenitors. Bone marrow and white blood cell samples from transplantedmice belonging to two independent experiments (Table 3) and harvested atdifferent time-points after transplant were analyzed as described insupplementary methods following the showed pipeline.

Overall, we mapped 5,173,892 sequencing reads on the transplanted mousegenome, resulting in 2,220 unique vector integration sites. The genomicdistribution of ISs from two independent experiments matched thepreviously reported LV preference for integration within transcriptionalunits (particularly within the first 50 Kb downstream of thetranscription start site -TSS-) (FIG. 16 a ), showing no skewing towardsany particular chromosome in the mouse genome (FIG. 16 b ). FIG. 16 ashows IS frequency distribution around TSS of the nearest RefSeq gene,spanning 500 Kb upstream and downstream the TSS. Numbers on the top arethe number of IS detected for all samples and time-points. FIG. 16 bshows chromosomal distribution of LV ISs in transplanted mice expressingthe EGFP transgene (black bars) or the coRPK therapeutic transgene (greybars), showing no skewing towards any particular chromosome.

Safety of the PGK-coRPK LV-based gene therapy was studied by clonalabundance estimations, calculating the percentage of sequence count foreach IS (a clonal mark) with respect to the total number of sequences ofthe dataset. Dot plots and heat map representations of the relativeabundance of each IS retrieved for each mouse (FIGS. 17, 18 and 19 )showed strong fluctuations in clonal composition of the different miceand of in vitro cultured Lin⁻ cells. FIG. 17 is a chart of trackedshared integrations between primary and secondary recipient micecarrying the therapeutic PGK-coRPK LV vector. Integrations detected ineither mouse in any organ and at any time are pooled. Secondaryrecipients received the pooled BM from transplanted mice coRPK11 to 14.The rest of the IS detected were detected in the primary or in thesecondary recipients. Numbers in the boxes show the representativenessin percentage of the corresponding integration in the referred mouse. Inaddition to ≥5% filter applied on integration analysis, all integrationswith a sequence count <3 were eliminated. FIG. 19 presents dot plotrepresentation of clonal abundance of pooled integrations in each mousein bone marrow. The relative percentage (y-axis) for each IS is relativeto the total number of sequences reads obtained in each dataset.Similarly to co-RPK transduced cells (FIG. 17 ), the graph indicatesthat the vast majority of transplanted mice show a polyclonal pattern ofhematopoietic repopulation.

Also, it was possible to appreciate that for several samples, a smallnumber of integrations contributed to a large amount of sequence reads(FIGS. 17 and 18 ), revealing a polyclonal pattern of repopulation oftransduced HSCs. In addition, tracked shared integration between primarymice carrying the therapeutic PGK-coRPK LV and subsequently transplantedsecondary mice showed no strong sharing of integrations between thegroups, confirming the absence of clonal dominance (FIG. 18 ).

To determine whether hallmarks of insertional mutagenesis were presentin transplanted mice, we assessed the occurrence of Common InsertionSites (CIS) similar to currently ongoing LV-mediated clinical trials.CIS are insertional hotspots that may result from integration bias atthe time of transduction or in vivo selection of clones harbouringvector integrations that confer growth advantage. CIS were identifiedusing an algorithm based on Abel and cols and the Grubbs test foroutliers finding no CIS and thus no alarming signs of genotoxicity bythis readout. Moreover, GO analysis revealed no skewing towards geneclasses involved in cancer, cell proliferation or regulation ofapoptosis in any of the integration datasets sorted bytissue-distribution, time point or abundance of repopulatinghematopoietic cell clones (FIG. 20 ). FIG. 20 represents LV genomicintegration profile. GO analysis was performed using the GREAT softwareon samples from transplanted mouse. All integrations retrieved from thisstudy (N=2220) showed overrepresentations of the gene functionsindicated on the left part of the figure. To address if the mostabundant integrations were enriched on specific gene classes, all ISswith a relative sequence count >5% of the entire dataset (shown in FIG.17 ) were selected, showing no GO gene classes overrepresented.

These results suggest neutrality of vector integration and demonstratethe safety of the PGK-coRPK LV in a preclinical setting.

Example 6 Human Clinical Trial

A clinical trial is conducted to evaluate safety and preliminaryefficacy of autologous hematopoietic stem cell transplantation (HSCT)using the EU/3/14/1130 medicinal product (autologous CD34+ hematopoieticstem cells transduced with the LV vector containing the RPK gene) inpatients with PKD with a history of severe and transfusion-dependentanemia refractory to splenectomy.

The ODD EU/3/14/1130 comprises a SIN LV vector expressing the coRPK gene(FIG. 21 ).

SIN LV vectors provide a more robust expression (Ellis 2005) and areless susceptible to transcriptional silencing than gamma-RV vectors(Pfeifer, Ikawa et al. 2002). They also show a much safer integrationprofile (Schroder, Shinn et al. 2002) (Mitchell, Beitzel et al. 2004)(Wu, Li et al. 2003), and because of the 400 bp deletion that they carryin the 3′ LTR sequence (Miyoshi, Blomer et al. 1998) (Zufferey, Dull etal. 1998), transgene expression is regulated by internal promoters,increasing the safety of the LV-based genetic modification.

The sequence of the accepted LV vector also includes severalmodifications to improve transgene expression and safety in targetcells.

One modification is the use of the human PGK promoter, alreadycharacterized by its stable in vivo activity and improved safetyproperties compared to other promoters used in gene therapy (Montini,Cesana et al. 2006, Modlich, Navarro et al. 2009, Montini, Cesana et al.2009, Biffi, Montini et al. 2013). Incorporation of the PGK promoterleads to a physiological expression of the transgene and a lowersusceptibility to transcriptional silencing (Gerolami, Uch et al. 2000,Zychlinski, Schambach et al. 2008).

Another modification is the coRPK gene to increase mRNA stability upontranscription. For the optimization the GeneArt® software has been used,increasing the GC content and removing cryptic splice sites in order toavoid transcriptional silencing and therefore increase transgeneexpression. The coRPK optimized sequence showed 80.4% homology with thehuman PKLR gene, with no changes in the amino acids sequence of theprotein.

Another modification is a mutated wPRE, lacking any residual openreading frame (Schambach, Bohne et al. 2006) is also included to improvethe level of expression and stability of the therapeutic gene. Thebackbone, promoter and wPRE* sequences of this LV vector (PGK-coRPK LV)are the same as the one corresponding to the medicinal product“Lentiviral vector containing the Fanconi anemia A (FANCA) gene for thetherapy of Fanconi anemia Type A patients” (Ref 141/2000), as well asvector backbone used in the currently ongoing clinical trial for themetachromatic leukodystrophy (MLD) (Biffi, Montini et al. 2013).

Mode of Action

After harvesting the CD34⁺ progenitor cells from PKD patients, eitherfrom bone marrow (BM) or mobilized peripheral blood cells, they aretransduced ex vivo with the medicinal product and the therapeutic vectorwill be integrated in the genome of the cells. Once integrated, thetherapeutic human gene (coRPK) are transcribed and translated withindeficient cells to produce the therapeutic RPK protein that is missingor reduced in PKD mature erythrocytes. Transduced PKD hematopoieticprogenitors are then genetically corrected, and thus able to produceRBCs with sufficient amounts of ATP to accomplish their functions (FIG.22 ). These genetically corrected hematopoietic progenitors (which willconstitute the medicinal product) are then transplanted back into thepatient, where once engrafted will generate normal erythrocytes,providing a life-long cure of the disease.

The active principle consists in a cellular suspension of correctedhematopoietic stem cells (CD34⁺) cells with the therapeutic LV vectorPGK.coRPK.wpre, designated as orphan drug by the European Commission(ODD EU/3/14/1130) for the treatment of PKD. Thus, this new medicamentshould be included in the groups of advanced therapy developments withinthe gene therapy sub-class.

The active principle is composed of a genetically modified cellularsuspension of at least 2×10⁶ CD34⁺ cells/kg of body weight with at least0.1 copies of the therapeutic vector per cell. The cells are suspendedin a saline buffer with 2% HSA.

The final therapeutic product is produced according to GMP rules, so theproduct requirements for its liberation and its infusion in the patientwill be related with the quality of the product. In relation to this,these specifications will include cellular viability ≥30%, sterility(Gram test and sterility by Pharmacopeia), absence of mycoplasma,absence of replication-competent LV particles, and demonstration of thepotency of the therapeutic potential by the detection of the presence ofat least 0.1 vector copies per cell by quantitative PCR. Additionally,investigation and research studies of the content of hematopoieticprogenitors and the vector copy number in these are performed. Threeindependent validations are performed with healthy control cells toassure the procedure reaches the above-described requirements.

The final product is packaged in a transfer bag sealed by heat andespecially for freezing and storage until its infusion to the patient;previously, samples will be collected for the precise correspondingquality controls.

Mobilization

Patients are mobilized in their respective hospitals, although the twofirst patients are mobilized in the Hospital del Niño Jesús, Madrid(Spain). Mobilization process includes in the administration ofrecombinant stimulating factor granulocyte colony (G-CSF, Neupogen,Amgen, Thousand Oaks, CA, USA) at doses of 12 mg/kg twice a day for upto eight days from birth, Plerixafor 4th day 240 mg/kg/d (Mozobil®,Genzyme Europe BV, Naarden, Netherlands) to four subcutaneous doses for4 consecutive days. The hematopoietic progenitor cells from peripheralblood are collected by leukapheresis, and large volumes from day 5 ofmobilization are sent through a cell separator according to standardprotocols of the Hospital Niño Jesús in Madrid. All the instruments andsolutions are CE marked and meet the specifications of the legislationfor medical devices.

CD34⁺ Cell Purification

Consistent with the mobilization process, the apheresis takes place inthe hospital where the patient is mobilized. Apheresis product isprocessed immediately to select hematopoietic progenitors (CD34⁺ cells)through MACS “magnetic cell sorting” technology (Miltenyi Biotec,Germany) which permits separation of cells by a high magnetic fieldgradient, through a powerful permanent magnet and a separation columnwith a ferromagnetic matrix. The CliniMACS (Miltenyi Biotec, BergischGladbach, Germany) system consists of a computer (CliniMACS®plusInstrument), a specific CD34⁺ selection software, a set of sterile tubes(CliniMACS Tubing Sets), magnetic-controlled reactive sterile instrument(CliniMACS CD34 Reagent) and a sterile buffer (CliniMACS PBS/EDTABuffer). The instrument and reagents used are CE marked and will meetthe specifications of the legislation for medical devices. During thisphase and in later washes nonspecific immunoglobulin (intravenousFlebogamma 5% 0.5 gr, Grifols) and human albumin (human albumin Grifols®20%, Grifols) are employed, which are later removed by washing aftercentrifugation. The CD34⁺ cells are then quantified. Microbiologicalcontrols on the products obtained will be performed by taking standardfungal, aerobic bacteria and anaerobes samples for cultures followingspecific protocols.

CD34⁺ Transduction

Transduction of the purified CD34⁺ cells with the ODD EU/3/14/1130 isperformed under GMP conditions in a time window within 48 h since theextraction of the cells from the patient (apheresis). Ex-vivo culture ofthe cells lasts less than 48 hours, and will be cultured followingstablished standards including the use of properly formulated mediaX-vivo-20 (Lonza), addition of hematopoietic growth factors (100 ng/mlhrSCF, 100 ng/ml hrFlt-3, 100 ng/ml TPO and 20 ng/mL IL-3 (all fromPrepotech), 1 μg/mL Pulmozyme and controlled 5% O₂ concentration.Transduction will be performed with a GMP LV batch of the ODDEU/3/14/1130 produced by VIVEbiotech (San Sebastian, Spain). Aftertransduction, cells are washed in X-vivo-20 (Lonza) to be finallypackaged in a transfer bag suitable for cryopreservation. Specificsamples are collected to determine if the final product meets all thespecifications already mentioned for its final liberation. Threeindependent validations are performed to evaluate the stability of theproduct. All products and solutions, including the vector, will meet thespecifications of the legislation for medical devices and clinical use.Before production, all raw materials (including consumables, biologicalreagents and chemical powders) will be inspected by the Quality Control(QC) Unit of CliniStem according to standard operating procedures(SOPs).

Conditioning

Patients are conditioned according to standardized and specificprotocols considered for the trial. To consider a patient forconditioning as an alternative, a back-up of 2×10⁶ unmanipulated CD34⁺cells/kg is kept frozen to be used in case the prepared product does notcompletely reconstitute the hematopoiesis of the treated patient.

Infusion

Before infusion, patient eligibility is checked to ensure they meet thestudy requirements. The day of infusion, premedication and prophylacticmedication used are recorded.

Example 7 Non-Clinical Development

Previous work demonstrated the feasibility of HSC gene therapy for PKDin mice when above 25% genetically corrected cells were transplanted.These results suggest that a significant number of donor gene-correctedHSCs (Zaucha, Yu et al. 2001) and high levels of transgene expressionare needed to achieve a therapeutic effect in PKD. We have developed anew therapeutic LV vector proposed for this clinical trial, harbouringthe hPGK eukaryotic promoter driving the expression of the PKLR cDNAthat was designated as an Orphan Drug on August 2014 (EU/3/14/1130).With this vector, we conducted a preclinical gene therapy protocol forPKD in a mouse model of the disease. With LV dosages based on clinicalstandards, ectopic RPK expression was able to normalize the erythroidcompartment, correcting the haematological phenotype and reverting organpathology. Metabolomic studies demonstrated the functional correction ofthe glycolytic pathway in genetically corrected RBCs, with no metabolicdisturbances observed in leukocytes. Remarkably, WBCs analyzed inparallel showed no alterations of the metabolic balance in leukocyteswhen RPK is ectopically expressed under the activity of an ubiquitouspromoter such as PGK, ruling out a leukocyte metabolic advantage aspossible safety concern and reinforces the therapeutic potential of theEU/3/14/1130 vector.

The multi-lineage reconstitution and the absence of any leukemic eventor clonal expansion in secondary recipients after the proliferativestress induced by BM re-transplant demonstrate the long-term stabilityand safety of the PGK-coRPK LV vector-based protocol. The use of thehuman PGK eukaryotic promoter that i) likely led to a more physiologicalexpression of the RPK transgene, ii) has been proven to be a weaktransactivator and iii) is being currently used in the clinical trialfor metachromatic leukodystrophy (MLD), could also account for thesafety of the whole procedure.

To assess the long-term safety of HSC gene therapy through the analysisof vector ISs, next generation sequencing was used to predict the riskof insertional oncogenesis in HSC. More than 5,173,892 sequences readswere mapped on the mouse genome to a total of 2220 unique vector IS,finding no evidence of in vivo expansion or selection of clonesharboring IS. Rather, our data shows the clonal composition and dynamicsof hematopoiesis after transplantation of transduced HSCs in mice,suggesting a genuine and stable genetic in vivo modification of HSC overtime. Overall, the analysis of the vector integration pattern emphasizesthe safety properties of the PGK-coRPK LV vector that provides PKDgenetic correction with no evidence of genotoxicity.

Example 8 Clinical Development

Protocol assistance for clinical studies is requested to a RegulatoryAgency. Our aim is to perform a clinical trial sponsored by the EuropeanCommission. The ForGeTPKD Consortium, composed by different cliniciansand basic researchers in Europe, has been established to focus in PKDresearch and development of new therapeutic strategies. The ForGeTPKDclinical trial will be the first administration of this medicinalproduct in humans. It is designed as an International, Multicenter,Phase I/II Open Label Study to evaluate the Safety and Efficacy ofTransplantation of Autologous CD34+ Cells Transduced Ex Vivo with aLentiviral vector containing the red-cell type Pyruvate Kinase (RPK)gene (EU/3/14/1130) in patients with Severe Pyruvate Kinase Deficiency.

Regulatory Status

The medicinal product has no marketing authorization at present time.The objective of the PKD Consortium is to move forward to the clinicaldevelopment of the medicinal product in order to eventually receive amarketing authorization.

The mentioned final product will be produced with a LV vector that hasreceived the Orphan Drug Designation related to:

Indication: Treatment of Pyruvate Kinase Deficiency

Criteria: The only curative treatment for PKD is allogeneic BMT, whichhas been used in patients with transfusion-dependent severe anemiarefractory to other measures. However, allogeneic BMT is not a widelyaccepted treatment for PKD (only one patient reported in the literature(Tanphaichitr, Suvatte et al. 2000)) as it is associated with severecomplications related to intensive pre-allo-BMT conditioning bychemotherapy or chemo-radiotherapy, as well as acute and chronicgraft-versus-host disease (GVHD). Our hypothesis is that gene therapyusing autologous hematopoietic stem cells transduced with viral vectorscontaining the wild-type version of the gene, provided by the ODDEU/3/14/1130, may represent a potential curative opportunity for thesepatients, avoiding the risks of GVHD, the main cause of failure of ahematopoietic progenitors transplant.

Active substance: Autologous CD34⁺ hematopoietic stem cells, transducedwith Lentiviral vector containing the red-cell type Pyruvate Kinase(RPK) gene (ODD EU/3/14/1130), expressing the wild type version of theprotein

Finished product: Frozen bag containing at least 2×10⁶ activesubstances/kg body weight of the patient, suspended in a saline bufferwith 2% HAS

Example 9 Pharmacology

Completed studies: The medicinal product developed includes severalmodifications in its sequence that provides some advantages for genetherapy of PKD: 1) The use of a SIN-LV vector design allowed arelatively easy and safe production of viral stocks, able to efficientlytransduce HSCs; 2) the use of a weak eukaryotic promoter such as hPGK,which is less susceptible to silencing by methylation (Gerolami, Uch etal. 2000), leads to physiological expression of the transgene, achievingtherapeutic levels with a viral dosage (1.65 VCN) within the clinicalstandards (Matrai, Chuah et al. 2010); and 3) the codon optimizedtransgene sequence and the presence of the mutated wPRE sequenceincreases transgene mRNA stability. No reporter gene was included in thetherapeutic vector sequence, avoiding possible immunogenic problems(Morris, Conerly et al. 2004); (Stripecke, Carmen Villacres et al.1999).

The developed hPGK-coRPK LV medicinal product efficiently reverted PKDpathology in both primary and secondary deficient mice transplanted withprogenitors transduced and corrected with the ODD EU/3/14/1130. Thecorrection was achieved with cells carrying on average 1.65 copies percell of the therapeutic transgene.

Human PGK promoter was potent enough to express clinically relevantlevels of coRPK protein, restoring the hemolytic phenotype intransplanted mice.

The genetic correction was able to: extend RBC half-life; normalize thehematological variables and reticulocyte levels; revert the compensatoryerythropoiesis constitutively activated in PKD mice; and rescue thepathology in spleen and liver, remarkably reducing the iron overload,which is one of the life-threatening complications of PKD. In addition,the ectopic expression of human RPK corrected the energetic defect inRBCs without altering the metabolic balance in WBCs, emphasizing theefficacy and safety of the medicinal product.

Example 10 Ongoing Studies

Transduction of human hematopoietic progenitors from healthy donors andPKD patients for the study of: 1) determining the efficiency oftransduction of the ODD EU/3/14/1130 in human cells; 2) defining theoptimal vector copy number/cell to get efficient and therapeuticexpression of the RPK therapeutic protein; and 3) defining the optimalconditions to get therapeutic transduction levels without losinghematopoietic stem cell ability.

Planned studies include set up of conditions for large-scaletransduction at the GMP facility, and pre-validation and 3 validationstudies to set up the optimal conditions to reach the requiredspecifications defined for the final therapeutic product.

Toxicology

Completed study results include: 1) The ectopic expression of human RPKcorrected the energetic defect in RBCs without altering the metabolicbalance in WBCs; 2) Genome integration analysis of the vector hasdemonstrated that (i) The analysis of the relative abundance of specificcell clones revealed an oligoclonal hematopoietic reconstitution forsome mice, with no clonal dominance for any primary and secondarytransplanted mice; (ii) CIS analysis, considered a hallmark ofinsertional mutagenesis, did not show any sign of genotoxicity, nor anabnormal enrichment of CIS over time, as the detected CIS from the twoindependent gene therapy experiments performed in mice were notrepresented by high sequence counts and did not preferentially targetoncogenes; (iii) GO analysis of the genes targeted by the LV integrationand analysis of the position of the vector integrations in specificregions of the genome demonstrated no skewing towards gene classesinvolved in cancer, cell proliferation or regulation of apoptosis; and(iv) Overall, medicinal product integration analysis did not show anyevidence of genotoxicity.

Planned studies include 1) Analysis of Recombinant CompetentLentiviruses (RCL) production: Human T lymphocytes from healthy donorsand from PKD patients will be transduced with the ODD EU/3/14/1130 andcultured in vitro for extended periods of time. Presence of the viralp24 protein will be analyzed in supernatants by ELISA to evaluate thepotential generation of RCLs and 2) Biodistribution of the medicinalproduct: Mouse hematopoietic progenitors will be transduced with the ODDEU/3/14/1130 and transplanted into lethally irradiated recipients. Onemonth post-transplant, animals will be sacrificed, and different organs(gonads, liver, kidney, brain, bone marrow, spleen and peripheral blood)will be analyzed for the presence of vector DNA; and 3) Vector integromein human cells: Hematopoietic progenitors from healthy donors and fromPKD patients will be transduced with the ODD EU/3/14/1130 andtransplanted into severe immunodeficient mice to allow the engraftmentand proliferation of human hematopoietic cells. At different time points(1, 2 and 3 months post-transplant) blood and BM transplants will betaken, sorted for human cells and subjected to vector integrome analysisas already performed with mouse cells.

Example 11 Human Clinical Trial

To test clinical efficacy, the ForGeTPKD trial will be conducted. Theproposed clinical trial aims to evaluate safety and preliminary efficacyof autologous HSCT using the EU/3/14/1130 medicinal product (autologousCD34⁺ hematopoietic stem cells transduced with Lentiviral vectorcontaining the red-cell type Pyruvate Kinase (RPK) gene) in patientswith PKD with a history of severe and transfusion-dependent anemiarefractory to splenectomy.

The primary objective is to evaluate treatment, safety, andtolerability/feasibility. The following endpoints will be measured inaccordance: 1) incidence and characterization of Adverse Events (AE),including: AE related to the infusion of the transduced cells, AEderived from conditioning prior to cell infusion, and AE derived fromclonal evolution related with the transduced cells; and 2) number ofpatients with stem cell engraftment at 30 days post-transplant.

Secondary objectives are to evaluate preliminary treatment efficacy. Thefollowing endpoints will be measured in accordance: 1) Number ofpatients who become “transfusion independent” at the end of the study,and the number of patients who still need transfusions after treatment;2) Ratio between the mean numbers of transfusions needed within thestudy period (1 year) with respect to the mean number of transfusions inthe last 1.5 years before baseline evaluation; 3) Clinically significantreduction of anemia, defined as the number of patients with a rise inhemoglobin levels in 2 gr/dL from baseline at the end of the study; 4)Clinically significant reduction of reticulocytosis, defined as thenumber of patients with a reduction of 50% from baseline evaluation atthe end of the study; and 5) Number of patients with stem cellengraftment where 1% transduced cells can be detected at 6 and 12 monthpost-cell infusion at the end of the study.

An exploratory objective is to evaluate treatment impact on patient'squality of life. The following end-point will be measured in accordance:Improvement in quality of life from baseline at the end of the study,using a quality of life questionnaire (SF-36 for adults or PEDSQL forchildren) and its translated validated versions in the language of theparticipant countries (Italian, Dutch and Spanish).

The ForGetPKD trial is a multicenter, international trial, which will becarried out in 3 EU member states: Spain, Italy and The Netherlands.Participating centers include Reference National Investigators andInstitutions for PKD diagnosis and treatment.

The trial will represent the first administration to humans of thedescribed product. It is designed as a non-comparative, open-label,single-dose, Phase I/II study.

Global Study duration will be 2 years from the first visit of the firstpatient to the last visit of the last patient. This includes 1 year ofrecruitment period and 1 year of treatment period and early (immediate)follow-up. After the end of the trial, included subjects will be askedto participate in a subsequent follow-up study that will monitor safetyand efficacy for up to 5 years after the transplant.

Study procedures include a Screening Period, a Treatment Period and aFollow-up Period. Details of visits on each phase and associated studyprocedures are detailed below and summarized in Table 4.

TABLE 4 Study Procedures Period Treatment Screening Period Period Followup period Visit −1 1 Pre- 0 Treatment 2 + 3 + 4 + 5 + Study proceduresscreening Baseline period 1 m 3 m 6 m 12 m Informed consent XInclusion/exclusion X X Criteria Medical History X Physical X X XExamination CBCs X X X X X X Biochemistry X X X X X X Coagulation X X XX X X Conditioning X regimen Transduced cells X infusion Discharge XBone Marrow X sampling Cell engraftment in X X X X peripheral bloodAverage vector X X X X copy number in peripheral blood Vectorintegration X X X X pattern in peripheral blood Cell engraftment in X Xbone marrow Average vector X X copy number in bone marrow Vectorintegration X X pattern in bone marrow Cell extraction X Availability ofX viable cells Quality of life X X questionnaire (SF- 36 or PEDSQL)Concomitant X X X X X X X Medication Adverse Events X X X X X X X

Screening Period Visit-1: Pre-Screening Visit

Potential candidates will be informed of the aims and characteristics ofthe trial, and written informed consent will be taken, fulfilled andsigned by the patient (or legal representative if underage), byduplicate. To be eligible for the study, patients must fulfill allinclusion criteria and none of the exclusion criteria, which will bechecked. This includes a pre-treatment procedure to mobilize and obtainviable CD34⁺ cells, to A. Store 2×10⁶ CD34+ cells/kg body weight toserve as a backup in case of non-engraftment, B. transduce at least6×10⁶ CD34+ cells/kg body weight with the EU/3/14/1130 vector togenerate the medicinal product and to perform all the quality controlneeded for the liberation of the medicinal product. Only patients withenough transduced cells available after liberation of the medicinalproduct (2×10⁶ transduced CD34⁺ cells/kg body weight) will be includedin the study.

The following procedures will be also performed in this visit:

-   -   Register of relevant medical o surgery history.    -   Register of demographic data and clinically relevant physical        examination findings    -   Register of relevant concomitant medications.    -   Peripheral blood testing for routine cell blood counts (CBC),        Biochemistry, Coagulation determination and serology.    -   Echocardiogram, Lung function test and thorax X-ray.    -   Quality of life questionnaire (SF-36 or PEDSQL).    -   Genetic diagnosis of PKD.

To be eligible for the study, patients have to fulfill all of thefollowing inclusion criteria and none of the exclusion criteria.

Inclusion criteria are male or female patients, Age >2 year old at thetime of recruitment, willing to give signed informed consent (which willbe signed by their parents or legal representative in case of childrenunder 18 years old), previous diagnosis for PKD confirmed by genetictesting, history of severe transfusion-dependent anemia, not responsiveto splenectomy, Candidate to Autologous Hematopoietic Stem CellTransplant, ≥2×10⁶ transduced CD34⁺ cells/kg body weight available, andtreated and followed for at least the past 2 years in a specializedcenter that maintained detailed medical records, including transfusionhistory.

Exclusion criteria are: positive for presence of human immunodeficiencyvirus type 1 or 2 (HIV 1 and HIV 2), uncorrected bleeding disorder,presence of other causes of hemolysis, any prior or current malignancyor myeloproliferative or immunodeficiency disorder, immediate familymember with a known or suspected Familial Cancer Syndrome (including butnot limited to hereditary breast and ovarian cancer syndrome, hereditarynon-polyposis colorectal cancer syndrome and familial adenomatouspolyposis), in patients with previous allogeneic transplant, presence ofresidual cells of donor origin, patients with severe complications thatafter medical evaluation are considered to suffer grade III/IV cardiac,pulmonary, hepatic or renal function abnormalities, uncontrolled seizuredisorder, diffusion capacity of carbon monoxide (DLco)<50% of predicted(corrected for hemoglobin), any other evidence of severe iron overloadthat, in the Investigator's opinion, warrants exclusion, participationin another clinical study with an investigational drug within 30 days ofScreening, availability of a HLA-identical family donor for allogeneicbone marrow transplant, pregnant or breast-feeding women, patients that,according to investigator criteria, will not be able to understand studypurposes, benefits and risks and/or to comply with study procedures, anpoor functional status, evidenced by a Karnofsky Index ≤80 in adults orLansky ≤80 in children.

The statistical analysis of the study will be descriptive. Qualitativeendpoints will be described by frequencies and percentages. Qualitativeendpoints include the adverse events, the number of patients with stemcell engraftment, the number of patients who become “transfusionindependent”, the number of patients who have a clinically significantreduction of anemia, the number of patients who have a clinicallysignificant reduction of reticulocytosis, the number of patients withstem cell engraftment where presence of transduced cells can bedetected, and the improvement in quality of life from baseline measuredby SF-36 or PEDSQL questionnaire. Quantitative endpoints will bedescribed by mean and standard deviation or by median and quartiles.Quantitative endpoints include the reductions of anemia andreticulocytosis from baseline, the number of transfusions needed withinthe study period with respect to the number of transfusion in the last 1year before baseline evaluation, and the vector copy number inperipheral blood and bone marrow. All endpoints will be described at theend of the study.

Previous work demonstrated the feasibility of HSC gene therapy for PKDin mice when above 25% genetically corrected cell were transplanted.These results suggest that a significant number of donor gene-correctedHSCs (Zaucha, Yu et al. 2001) and high levels of transgene expressionare needed to achieve a therapeutic effect in PKD. We have developed anew therapeutic LV vector proposed for this clinical trial, harboringthe hPGK eukaryotic promoter driving the expression of the PKLR cDNAthat was designated as Orphan Drug on August 2014 (EU/3/14/1130). Withthis vector we conducted a preclinical gene therapy protocol for PKD ina mouse model of the disease. With LV dosages based on clinicalstandards, ectopic RPK expression was able to normalize the erythroidcompartment, correcting the hematological phenotype and reverting organpathology. Metabolomic studies demonstrated the functional correction ofthe glycolytic pathway in genetically corrected RBCs, with no metabolicdisturbances observed in leukocytes. Remarkably, WBCs analyzed inparallel showed no alterations of the metabolic balance in leukocyteswhen RPK is ectopically expressed under the activity of an ubiquitouspromoter such as PGK, ruling out a leukocyte metabolic advantage aspossible safety concern and reinforces the therapeutic potential of theEU/3/14/1130 vector.

The multi-lineage reconstitution and the absence of any leukemic eventor clonal expansion in secondary recipients after the proliferativestress induced by BM re-transplant demonstrate the long-term stabilityand safety of the PGK-coRPK LV vector-based protocol. The use of thehuman PGK eukaryotic promoter that likely led to a more physiologicalexpression of the RPK transgene, that has been proven to be a weaktransactivator and is being currently used in the clinical trial formetachromatic leukodystrophy (MLD) could also account for the safety ofthe whole procedure.

To assess the long-term safety of HSC gene therapy through the analysisof vector ISs, next generation sequencing was used to predict the riskof insertional oncogenesis in HSC. More than 5,173,892 sequences readswere mapped on the mouse genome to a total of 2220 unique vector ISs,finding no evidence of in vivo expansion or selection of clonesharboring IS. Rather, our data show the clonal composition and dynamicsof hematopoiesis after transplantation of transduced HSCs in mice,suggesting a genuine and stable genetic in vivo modification of HSC overtime. Overall, the analysis of the vector integration pattern emphasizesthe safety properties of the PGK-coRPK LV vector that provides PKDgenetic correction with no evidence of genotoxicity.

The invention may be further defined by reference to the followingillustrative clauses:

1. An expression cassette comprising a polynucleotide sequencecomprising in the following 5′ to 3′ order:

-   -   a) a promoter sequence;    -   b) a sequence encoding a gene product; and    -   c) an ribonucleic acid (RNA) export signal,    -   wherein the promoter sequence is operably linked to the sequence        encoding the gene product.

2. The expression cassette of clause 1, wherein the promoter is aphosphoglycerate kinase (PGK) promoter.

3. The expression cassette of clause 1 or clause 2, wherein the geneproduct is a therapeutic gene product.

4. The expression cassette of clause 3, wherein the therapeutic geneproduct is a pyruvate kinase (PK) polypeptide, optionally a pyruvatekinase, liver and red blood cell (PKLR) polypeptide.

5. The expression cassette of any of clauses 1-4, wherein the sequenceencoding the gene product is codon-optimized.

6. The expression cassette of clause 5, wherein the codon-optimizedsequence comprises a sequence having at least 85% identity to SEQ ID NO:8.

7. The expression cassette of any of clauses 1-6, wherein the RNA exportsignal is a mutated post-transcriptional regulatory element of thewoodchuck hepatitis virus (wPRE).

8. The expression cassette of clause 7, wherein the mutated wPRE is achimeric wPRE comprising a sequence having at least 80% identity to SEQID NO:24.

9. The expression cassette of any of clauses 1-8, further comprising oneor more enhancer sequences.

10. The expression cassette of any of clauses 1-9, further comprising apolypurine tract (PPT) or polyadenylation (polyA) signal sequence.

11. The expression cassette of any of clauses 1-10, further comprisingone or more of the following sequences:

-   -   i) a packing signal sequence;    -   ii) a truncated Gag sequence;    -   iii) a Rev responsive element (RRE);    -   iv) a central polypurine tract (cPPT);    -   v) a central terminal sequence (CTS); and    -   vi) an upstream sequence element (USE), optionally from simian        virus 40 (SV40-USE).

12. The expression cassette of any one of clauses 1-11, furthercomprising 5′ and 3′ long terminal repeat sequences.

13. A recombinant gene delivery vector comprising the expressioncassette of any one of clauses 1-12.

14. The recombinant gene delivery vector of clause 13, wherein therecombinant gene delivery vector is a virus or viral vector.

15. The recombinant gene delivery vector of clause 14, wherein the virusor viral vector is a lentivirus (LV).

16. A cell comprising the expression cassette of any of clauses 1-12 orthe recombinant gene delivery vector of any of clauses 13-15.

17. The cell of clause 16, wherein the cell is a hematopoietic stemcell.

18. The cell of clause 16, wherein the cell is a committed hematopoieticerythroid progenitor cell.

19. A pharmaceutical composition comprising a pharmaceuticallyacceptable excipient and the recombinant gene delivery vector of any ofclauses 13-15 or the cell of any of clauses 16-18.

20. A method of treating or preventing a disease or disorder in asubject in need thereof, comprising providing to the subject thepharmaceutical composition of clause 19.

21. The method of clause 20, wherein the disease or disorder is aPyruvate Kinase Deficiency (PKD) and the gene product is a pyruvatekinase (PK) polypeptide, optionally a pyruvate kinase, liver and redblood cell (PKLR) polypeptide.

22. The method of clause 20 or clause 21, wherein the pharmaceuticalcomposition comprises the recombinant gene delivery vector.

23. The method of clause 20 or clause 21, wherein the pharmaceuticalcomposition comprises the cell.

24. The method of clause 23, wherein the cell is autologous to thesubject.

25. A method for expressing a transgene in erythroid cells, comprisingcontacting one or more erythroid cells with an effective amount of arecombinant viral vector, wherein the vector comprises a humanphosphoglycerate kinase promoter, a codon optimized version of a humanpyruvate kinase, liver and red blood cell (PKLR) cDNA transgene, and amutated post-transcriptional regulatory element of the woodchuckhepatitis virus, wherein following said contacting, PKLR is expressed atdetectable levels in the one or more erythroid cells.

1. An expression cassette for pyruvate kinase deficiency (PKD),comprising a polynucleotide sequence comprising in the following 5′ to3′ order: a) a promoter sequence; b) a codon-optimized sequence encodinga human PKLR gene product; and c) a mutated post-transcriptionalregulatory element of woodchuck hepatitis virus (wPRE); wherein themutated wPRE is a chimeric wPRE comprising a sequence having at least90% identity to SEQ ID NO:24; wherein the promoter sequence is operablylinked to the codon-optimized sequence encoding a human PKLR geneproduct, and wherein the codon-optimized sequence encoding a human PKLRgene product shares at least 90% identity to SEQ ID NO:
 8. 2. Theexpression cassette of claim 1, wherein the promoter is aphosphoglycerate kinase (PGK) promoter.
 3. (canceled)
 4. (canceled) 5.The expression cassette of claim 1, further comprising a polypurinetract (PPT).
 6. The expression cassette of claim 1, further comprising apolyadenylation (polyA) signal sequence.
 7. The expression cassette ofclaim 1, wherein the expression cassette shares at least 95% identity toSEQ ID NO:
 26. 8. The expression cassette of claim 7, wherein theexpression cassette shares at least 99% identity to SEQ ID NO:
 26. 9. Arecombinant gene delivery vector comprising the expression cassette ofclaim 1, wherein the recombinant gene delivery vector is a lentiviralvector.
 10. The recombinant gene delivery vector of claim 9, comprisingthe following sequences in 5′ to 3′ order: a) a 5′ LTR, optionally amodified 5′ LTR; b) an HIV-1 w sequence; c) an RRE; d) a cPPT/CTSsequence; e) a PGK promoter sequence, optionally a human PGK promotersequence; f) a codon-optimized sequence encoding a human PKLR geneproduct having at least 90% identity to SEQ ID NO: 8; g) a mutant wPREsequence having at least 90% identity to SEQ ID NO:24; i) a modified 3′LTR; j) an SV40 poly(A) signal; k) an SV40 origin of replication (ori);u) a CMV enhancer sequence; and v) a CMV promoter sequence.
 11. Therecombinant gene delivery vector of claim 9, wherein the recombinantgene delivery vector is a therapeutic gene delivery vector.
 12. Therecombinant gene delivery vector of claim 11, wherein the recombinantgene delivery vector is capable of long-term stability and safety inclinical use.
 13. A cell comprising the recombinant gene delivery vectorof claim
 9. 14. The cell of claim 13, wherein the cell is ahematopoietic stem cell.
 15. The cell of claim 14, wherein the cell is acommitted hematopoietic erythroid progenitor cell.
 16. A pharmaceuticalcomposition comprising a pharmaceutically acceptable excipient and thecell of claim
 13. 17. A method of treating or preventing Pyruvate KinaseDeficiency (PKD) in a subject in need thereof, comprising providing tothe subject the pharmaceutical composition of claim
 16. 18.-19.(canceled)
 20. The method of claim 17, wherein the cell is autologous tothe subject.
 21. A method for expressing a human PKLR gene in erythroidcells, comprising contacting one or more erythroid cells with aneffective amount of a recombinant viral vector, wherein the vectorcomprises a human phosphoglycerate kinase promoter, a codon-optimizedPKLR transgene, and a mutated post-transcriptional regulatory element ofthe woodchuck hepatitis virus, wherein the codon-optimized PKLRtransgene shares at least 90% identity with SEQ ID NO: 8 and the mutatedpost-transcriptional regulatory element of the woodchuck hepatitis virusshares at least 90% identity with SEQ ID NO: 24, and wherein followingsaid contacting, PKLR is expressed at detectable levels in the one ormore erythroid cells.